FoxO1 Suppresses Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication and Controls Viral Latency

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) has latent and lytic replication phases, both of which contribute to the development of KSHV-induced malignancies. Among the numerous factors identified to regulate the KSHV life cycle, oxidative stress, caused by imbalanced clearing and production of reactive oxygen species (ROS), has been shown to robustly disrupt KSHV latency and induce viral lytic replication. In this study, we identified an important role of the antioxidant defense factor forkhead box protein O1 (FoxO1) in the KSHV life cycle. Either chemical inhibition of the FoxO1 function or knockdown of FoxO1 expression led to an increase in the intracellular ROS level that was subsequently sufficient to disrupt KSHV latency and induce viral lytic reactivation. On the other hand, treatment with N-acetyl-l-cysteine (NAC), an oxygen free radical scavenger, led to a reduction in the FoxO1 inhibition-induced ROS level and, ultimately, the attenuation of KSHV lytic reactivation. These findings reveal that FoxO1 plays a critical role in keeping KSHV latency in check by maintaining the intracellular redox balance.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several cancers, including Kaposi's sarcoma (KS). Both the KSHV latent and lytic replication phases are important for the development of KS. Identification of factors regulating the KSHV latent phase-to-lytic phase switch can provide insights into the pathogenesis of KSHV-induced malignancies. In this study, we show that the antioxidant defense factor forkhead box protein O1 (FoxO1) maintains KSHV latency by suppressing viral lytic replication. Inhibition of FoxO1 disrupts KSHV latency and induces viral lytic replication by increasing the intracellular ROS level. Significantly, treatment with an oxygen free radical scavenger, N-acetyl-l-cysteine (NAC), attenuated the FoxO1 inhibition-induced intracellular ROS level and KSHV lytic replication. Our works reveal a critical role of FoxO1 in suppressing KSHV lytic replication, which could be targeted for antiviral therapy.

FIG 1

FoxO1 inhibitor AS1842856 disrupts KSHV latency and induces KSHV lytic replication. (A, B) Fluorescence microscopy images of EGFP-positive cells among iSLK-RGB-BAC16 cells treated with 0 (DMSO alone), 5, 7, and 10 μM FoxO1 inhibitor AS1842856 for 48 h. Representative images are shown (A), and the results of statistical analysis from 3 repeats, each with 5 images, are presented (B). Cells treated with 200 μM H₂O₂, 1 and 3 mM sodium butyrate (NaB), and 1 μl/ml of doxycycline (Dox) were used as controls. (C) Flow cytometry analysis of EGFP expression in iSLK-RGB-BAC16 cells treated with 0, 5, 7, and 10 μM FoxO1 inhibitor AS1842856 for 72 h. Cells treated with 0 μM FoxO1 inhibitor (DMSO) were used as negative controls. Cells treated with 200 μM H₂O₂, 1 and 3 mM NaB, and 1 μl/ml of Dox were used as positive controls. Comp-GFP-A indicates relative EGFP intensity. **, P < 0.01.

FIG 2

FoxO1 inhibitor AS1842856 induces expression of KSHV lytic genes. (A to E) Reverse transcription-quantitative real-time PCR (RT-qPCR) analysis of viral lytic transcripts RTA (A), PAN RNA (B), MTA (C), ORF-K8 (D), and ORF65 (E) in iSLK-RGB-BAC16 cells treated with 0 (DMSO alone), 5, 7, and 10 μM FoxO1 inhibitor AS1842856 for 48 h. The values for untreated cells (cells treated with DMSO alone [0 μM FoxO1 inhibitor]) were set equal to 1 as a reference. Cells treated with 200 μM H₂O₂, 1 and 3 mM sodium butyrate (NaB), and 1 μl/ml of doxycycline (Dox) were used as positive controls. (F) Western blot analysis for the expression of KSHV lytic proteins RTA, ORF-K8, MTA, and ORF65 in iSLK-RGB-BAC16 cells treated with 0, 5, 7, and 10 μM the FoxO1 inhibitor AS1842856 for 72 h. Cells treated with 200 μM H₂O₂, 1 and 3 mM sodium butyrate (NaB), and 1 μl/ml of doxycycline (Dox) were used as positive controls. β-Actin was used as a loading control. *, P < 0.05; **, P< 0.01; ***, P < 0.001; NS, not significant.

FIG 3

Knockdown of FoxO1 disrupts KSHV latency and induces viral lytic replication. (A) Detection of the FoxO1, FoxO3a, and FoxO4 proteins in iSLK-RGB-BAC16 cells treated with 0 (DMSO alone), 5, 7, and 10 μM FoxO1 inhibitor AS1842856 for 48 h. (B, C) Knockdown of FoxO1 with two independent siRNAs in iSLK-RGB-BAC16 cells verified by RT-qPCR (B) and Western blotting (C). Cells transfected with a scrambled siRNA (Scrambled) and two independent siRNAs targeting FoxO1 (siFoxO1-1 and siFoxO1-2) for 48 h were examined by Western blotting. (D, E) Fluorescence microscopy images of EGFP-positive cells in iSLK-RGB-BAC16 cells following knockdown of FoxO1 with two independent siRNAs for 72 h. Representative images are shown (D), and the results of statistical analysis from 3 repeats, each with 5 images, are presented (E). Cells treated with 1 μl/ml of doxycycline (Dox) for 72 h were used as positive controls. (F) Flow cytometry analysis of EGFP expression in iSLK-RGB-BAC16 cells following knockdown of FoxO1 with two independent siRNAs for 72 h. Cells treated with 1 μl/ml of Dox for 72 h were used as controls. (G, H) Reverse transcription quantitative real-time PCR (RT-qPCR) analysis of the viral lytic transcripts RTA (G) and ORF65 (H) in iSLK-RGB-BAC16 cells following knockdown of FoxO1 with two independent siRNAs for 48 h. Cells treated with 1 μl/ml of Dox for 48 h were used as positive controls. (I) Western blot analysis for the expression of KSHV lytic protein ORF-K8 in iSLK-RGB-BAC16 cells following knockdown of FoxO1 with two independent siRNAs for 72 h. Cells treated with 1 μl/ml of Dox for 48 h were used as positive controls. The numbers under the ORF-K8 Western blot are the relative expression levels of ORF-K8 using β-actin as a loading control. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

Inhibition of FoxO1 triggers KSHV lytic replication by inducing intracellular ROS. (A) The FoxO1 inhibitor AS1842856 increases the intracellular ROS level in iSLK-RGB-BAC16 cells. Cells were treated with 0 (DMSO alone), 5, 7, and 10 μM the FoxO1 inhibitor AS1842856 for 48 h and analyzed by flow cytometry for intracellular ROS level. Cells treated with 0 μM FoxO1 inhibitor (DMSO) were used as negative controls. Cells treated 200 μM H₂O₂ with were used as positive controls. (B) The H₂O₂ scavenger NAC antagonizes the effect of FoxO1 inhibition. iSLK-RGB-BAC16 cells were treated with 5 μM the FoxO1 inhibitor AS1842856 or 200 μM H₂O₂ with or without 10 mM NAC and analyzed by flow cytometry for intracellular ROS levels. (C) NAC antagonizes the KSHV lytic replication induced by the FoxO1 inhibitor AS1842856. iSLK-RGB-BAC16 cells were treated with 5, 7, and 10 μM the FoxO1 inhibitor AS1842856 or 200 μM H₂O₂ with or without 10 mM NAC and analyzed for EGFP expression by flow cytometry. Cells treated with 0 μM FoxO1 inhibitor (DMSO) were used as negative controls. (D) NAC antagonizes the KSHV lytic replication induced by FoxO1 knockdown. iSLK-RGB-BAC16 cells were transfected with two independent siRNAs targeting FoxO1 (siFoxO1-1 and siFoxO1-2) with or without 10 mM NAC for 48 h and analyzed for EGFP expression by flow cytometry. Cells treated with 200 μM H₂O₂ were used as positive controls.