Fine-Tuning of the Kaposi’s Sarcoma-Associated Herpesvirus Life Cycle in Neighboring Cells through the RTA-JAG1-Notch Pathway

<div><p>Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is an oncogenic pathogen that displays latent and lytic life cycles. In KS lesions, infiltrated immune cells, secreted viral and/or cellular cytokines, and hypoxia orchestrate a chronic pro-lytic microenvironment that can promote KSHV reactivation. However, only a small subset of viruses spontaneously undergoes lytic replication in this pro-lytic microenvironment while the majority remains in latency. Here, we show that the expression of the Notch ligand JAG1 is induced by KSHV-encoded replication and transcription activator (RTA) during reactivation. JAG1 up-regulation activates Notch signaling in neighboring cells and prevents viral lytic replication. The suppression of JAG1 and Notch1 with inhibitors or small interfering RNA promotes lytic replication in the presence of RTA induction or under conditions of hypoxia. The underlying mechanism involves the Notch downstream effector hairy and enhancer of split 1 (Hes1), which directly binds lytic gene promoters and attenuates viral lytic gene expression. RTA interacts with lymphoid enhancer-binding factor 1 (LEF1), disrupts LEF1/Groucho/TLE suppressive complexes and releases LEF1 to activate JAG1 expression. Taken together, our results suggest that cells with viral lytic replication can inhibit KSHV reactivation in neighboring cells through an RTA-JAG1-Notch pathway. These data provide insight into the mechanism by which the virus maintains the balance between lytic and latent infection in the pro-lytic tumor microenvironment.</p></div

RTA induced JAG1 activates Notch signaling in neighboring cells through direct interaction.

<p>(A) Schematic illustrating the co-culture assay. SLK-Ctrl cells (0.4 million cells) or SLK-RTA cells (0.4 million cells) were co-cultured with iSLK.RGB (0.4 million cells) in 100 mm dish. At 24 and 48 h post co-culture, the RFP positive iSLK.RGB cells were sorted by FACS, followed by quantification of Hes1 and Hey1 expression by qPCR (B, C) or RTA, ICN1, Hes1 and JAG1 expression by western blotting (D). (E) Schematic illustrating the co-culture assays to detect lytic gene expression. SLK-Ctrl cells (0.4 million cells) or SLK-RTA cells (0.4 million cells) were co-cultured with iSLK.RGB (0.4 million cells) in 100 mm dish for 24 h and cells were treated with a low dose of doxycycline (100 ng/ml) to induce reactivation for 36 h. RFP and GFP double positive iSLK.RGB cells were sorted by FACS, followed by quantification of lytic gene expression by qPCR (F) or RTA, ICN1, Hes1 and JAG1 expression by western blotting (G). Data were expressed as the mean ± s.e.m., n = 3, *p<0.05, **p<0.01, ***p<0.001.</p

RTA up-regulates Notch components in different cell types.

<p>(A–C) iSLK.RGB cells, TRE-BCBL1-RTA cells, and iSLK cells were plated in 6-well plates at 0.1 million cells per well. After 24 h, the cells were induced with doxycycline (5 μg/ml) for 24 h and the expression of Notch components was measured by qPCR and western blotting. The data were normalized to GAPDH expression. (D) HEK293T cells seeded in 6-well plates were transfected with RTA (4 μg) using Lipofectamine 2000 for 24 h and the expression of Notch components was measured by qPCR and western blotting. (E) Immunofluorescence imaging of iSLK cells treated with or without doxycycline for 24 h. The JAG1 (Green) and RTA (Red) in the nucleus were labeled with the indicated primary and secondary antibodies. Scale bars represent 20 μm. (F) iSLK.RGB cells, TRE-BCBL1-RTA cells and iSLK cells were plated in 6-well plates at 0.1 million cells per well and treated with or without doxycycline for 24 h. HEK293T cells were transfected with RTA (4 μg) using Lipofectamine 2000 for 24 h in 6-well plates. The protein level of ICN1 was quantified by western blotting. (G) Hes1 and Hey1 were quantified by qPCR. The data were normalized to GAPDH expression. Data were expressed as the mean ± s.e.m., n = 3, *p<0.05.</p

Hes1 inhibits RTA-induced activation of KSHV lytic genes.

<p>(A) HEK293T cells were plated into 12-well plates at 0.5 million cells per well, a dual luciferase assay was performed in HEK293T cells transiently transfected with expression vectors containing RTA (1 μg), various reporter plasmids containing lytic gene promoters (100 ng), and increasing amounts of Hes1 (250 ng, 500ng or 1 μg) using Lipofectamine 2000. Total transfected DNA was normalized with pcDNA3.1. (B, C) A dual luciferase assay was performed in HEK293T cells transiently transfected with different vector combinations. (B) Mutant K8 (Left) or mutant ORF59 (Right) promoters were transfected with RTA and increasing amounts of Hes1. (C) Mutant Hes1 or wild type Hes1 containing plasmids were transfected with RTA and K8 or ORF59 promoters. Data were expressed as the mean ± s.e.m., n = 3, *p<0.05, **p<0.01, ***p<0.001.</p

RTA interacts with transcription factors downstream of the Wnt pathway to up-regulate JAG1.

<p>(A) A dual luciferase assay was performed in HEK293T cells plated in 12-well plates co-transfected with RTA or vector plasmids (2 μg each) and Wnt reporter plasmids TOP/FOPFlash (0.2 μg). LiCl (20mM) treatment was used as positive control. (B, C) Quantification of JAG1 expression by western blotting and qPCR in iSLK cells (70–80% confluence) treated with doxycycline (5 μg/ml) plus DMSO or salinomycin for (2μM) 24 h in 6-well plates. (D, E) JAG1 expression was quantified in iSLK cells transfected with siRNA (200 pM) against LEF1 (siLEF1) or scramble control (siCtrl) for 8 h when cells reached 60% confluence and treated with doxycycline for another 24 h. (F) JAG1 protein levels were quantified by western blotting in HEK293T cells transfected with RTA-SF, RTA-ΔSTAD or vector plasmids (4 μg each). (G, I, J) Co-immunoprecipitation assays were performed in HEK293T cells plated in 100 mm dish to test the physical interactions between SF-RTA and HA-LEF1 (12 μg each) (G), SF-RTA-ΔSTAD and HA-LEF1 (12 μg each) (I), and SF-TLE2 and HA-LEF1 (12 μg each) (J). (H) Immunofluorescence assay was performed in HEK293T cells transfected with SF-RTA and HA-LEF1 plasmids. Green color indicates LEF1 expression, Red color indicates RTA expression. Scale bars represent 10 μm. (K) A competition co-immunoprecipitation assay was performed in HEK293T cells plated in 100 mm dish to test RTA disrupted the LEF1/TLE2 complex. SF-TLE2 (4 μg) was co-transfected with HA-LEF1 (4 μg) alone or co-transfected with HA-LEF1 (4 μg) and increasing amounts of HA-RTA (4 and 8 μg). Precipitated HA-LEF1 was analyzed by western blotting.</p

Knockdown of JAG1 or Notch1 up-regulates lytic gene expression.

<p>(A, C) Quantification of the expression of the indicated lytic genes in iSLK.RGB cells transfected with siRNA (100 pM per well) against JAG1 (siJAG1) or scramble controls (siCtrl) in 6-well plates for 8 h when cells reached 60% confluence and treated with (A) or without doxycycline (C) for another 24 h. (B, D) Quantification of the expression of the indicated lytic genes in iSLK.RGB cells transfected with siRNA (100 pM per well) against Notch1 (siNotch1) or scramble controls (siCtrl) and treated with (B) or without doxycycline (D). Values represent the mean ± s.e.m., n = 3, *p<0.05, **p<0.01, ***p<0.001. (E–H) The efficiency of JAG1 or Notch1 depletion was detected by western blotting (E, F) and qPCR (G, H).</p

Hes1 inhibits RTA self-transactivation.

<p>(A) Schematic illustrating the N-box Hes1 binding motifs within the RTA promoter. (B) Primers were designed to cover the RTA promoter. (C, D) 293.219 cells plated in 100 mm dish at 4 million cells 24 h before transfection. HA-Hes1 and control plasmids (12 μg) were transfected and ChIP assay was performed 24 h after transfection. Extracts were subjected to immunoprecipitation with an anti-HA-tag antibody and purified DNA elute was quantified by gel analysis (C) or qPCR with the indicated primers from B (D). (E) Hes1 represses the transcriptional activity of the RTA promoter. Dual luciferase assay was performed in HEK293T cells plated in 12-well plates at 0.5 million per well 24 h after transfection. The cells were transiently transfected with expression vectors containing RTA (1 μg), reporter plasmids containing full length RTA promoter or RTA promoter truncations (100 ng), and increasing amounts of Hes1 (250 ng, 500 ng or 1μg) using Lipofectamine 2000. Total transfected DNA was normalized with pcDNA3.1. Data were expressed as the mean ± s.e.m., n = 3, *p<0.05, **p<0.01, ***p<0.001.</p

A proposed working model.

<p>RTA up-regulates JAG1 expression by disrupting LEF1/TLE suppressive complex via competitively binding with LEF1. Elevated membrane-bound JAG1 in RTA expressing cells interacts with Notch receptors and subsequently activates Notch signaling by producing higher level of Notch Intracellular Domain (ICN) in neighboring cells. Notch downstream effector Hes1 is up-regulated in response to activated Notch signaling. Hes1 directly binds and suppresses RTA and lytic gene promoters which in turn inhibits KSHV reactivation in the context of a specific microenvironment that drives lytic replication.</p

Herpesviral G Protein-Coupled Receptors Activate NFAT to Induce Tumor Formation via Inhibiting the SERCA Calcium ATPase

<div><p>G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi’s sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of “constitutive” NFAT activation by viral GPCRs.</p></div

Study selection process and results.

Study selection process and results.</p