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. 2018 Jan 16;9(1):e02217-17.
doi: 10.1128/mBio.02217-17.

Deregulation of HDAC5 by Viral Interferon Regulatory Factor 3 Plays an Essential Role in Kaposi's Sarcoma-Associated Herpesvirus-Induced Lymphangiogenesis

Hye-Ra Lee  1 , Fan Li  2 , Un Yung Choi  3 , Hye Ryun Yu  4 , Grace M Aldrovandi  5 , Pinghui Feng  3 , Shou-Jiang Gao  3 , Young-Kwon Hong  6 , Jae U Jung  7
Affiliations

Deregulation of HDAC5 by Viral Interferon Regulatory Factor 3 Plays an Essential Role in Kaposi's Sarcoma-Associated Herpesvirus-Induced Lymphangiogenesis

Hye-Ra Lee et al. mBio. .

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi's sarcoma (KS), which is one of the most common HIV-associated neoplasms. The endothelium is the thin layer of squamous cells where vascular blood endothelial cells (BECs) line the interior surface of blood vessels and lymphatic endothelial cells (LECs) are in direct contact with lymphatic vessels. The KS lesions contain a prominent compartment of neoplastic spindle morphology cells that are closely related to LECs. Furthermore, while KSHV can infect both LECs and BECs in vitro, its infection activates genetic programming related to lymphatic endothelial cell fate, suggesting that lymphangiogenic pathways are involved in KSHV infection and malignancy. Here, we report for the first time that viral interferon regulatory factor 3 (vIRF3) is readily detected in over 40% of KS lesions and that vIRF3 functions as a proangiogenic factor, inducing hypersprouting formation and abnormal growth in a LEC-specific manner. Mass spectrometry analysis revealed that vIRF3 interacted with histone deacetylase 5 (HDAC5), which is a signal-responsive regulator for vascular homeostasis. This interaction blocked the phosphorylation-dependent cytosolic translocation of HDAC5 and ultimately altered global gene expression in LECs but not in BECs. Consequently, vIRF3 robustly induced spindle morphology and hypersprouting formation of LECs but not BECs. Finally, KSHV infection led to the hypersprouting formation of LECs, whereas infection with a ΔvIRF3 mutant did not do so. Collectively, our data indicate that vIRF3 alters global gene expression and induces a hypersprouting formation in an HDAC5-binding-dependent and LEC-specific manner, ultimately contributing to KSHV-associated pathogenesis.IMPORTANCE Several lines of evidences indicate that KSHV infection of LECs induces pathological lymphangiogenesis and that the results resemble KS-like spindle morphology. However, the underlying molecular mechanism remains unclear. Here, we demonstrated that KSHV vIRF3 is readily detected in over 40% of various KS lesions and functions as a potent prolymphangiogenic factor by blocking the phosphorylation-dependent cytosolic translocation of HDAC5, which in turn modulates global gene expression in LECs. Consequently, vIRF3-HDAC5 interaction contributes to virus-induced lymphangiogenesis. The results of this study suggest that KSHV vIRF3 plays a crucial role in KSHV-induced malignancy.

Keywords: Kaposi's sarcoma-associated herpesvirus; angiogenesis; histone deacetylase.

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Figures

FIG 1
FIG 1
vIRF3 expression in KS tissues. We obtained KS tissue microarrays from AIDS Cancer Specimen Resource and performed immunohistochemistry, visualized by Aperio F.L. digital pathological scanning. A biopsy sample of KS TMA was stained with anti-LANA and anti-vIRF3 (CM-A807). Representative images show the LANA staining of skin KS lesion and the vIRF3 staining of skin, tonsil, and mouth KS lesions and positive staining corresponding to either LANA or vIRF3 in total KS biopsy specimens embedded in TMA.
FIG 2
FIG 2
vIRF3 expression facilitates sprouting formation in a LEC-specific manner. (A) Induction of spindle morphogenesis by vIRF3 in LECs but not in BECs (left panel) and real-time RT-PCR measurement of levels of podoplanin, Prox1, Neutropilin-1, and CXCR4 from LEC-E6/E7 and BEC-E6/E7 cells (middle panel). Prox1 expression was detected in LEC-E6/E7 cells (right panel). IB, immunoblot. (B) Tube formation assay. Either BECs expressing vector and vIRF3 or LECs expressing vector and vIRF3 were tested. The assay was monitored at the indicated time points. (C) Sprouting formation assay. Assays were performed on matrigel with LEC-E6/E7 vector and LEC-E6/E7 V5/vIRF3.
FIG 3
FIG 3
vIRF3 is required for KSHV-induced tube formation and sprouting formation. (A) (Left panel) Construction of KSHV ΔvIRF3 BAC16. A schematic diagram of KSHV ΔvIRF3 BAC16 construction is shown. (Right panel) Wild-type (WT), ΔvIRF3, and R-ΔvIRF3 recombinant KSHV-infected primary LECs were harvested at 72 h postinfection, and equal amounts of cell lysates were subjected to SDS-PAGE. (Right panel) Immunoblotting (IB) was performed with the indicated antibodies. (B and C) KSHV-induced tube formation and sprouting formation. (Left panel) WT, ΔvIRF3, and R-ΔvIRF3 recombinant KSHV-infected primary LECs were placed in Matrigel to perform sprouting formation and tube formation assays for the indicated times. (Right panel) Total tube lengths formed during the assay were measured by tube formation ACAS image analysis. *, P < 0.05. "MOCK" indicates KSHV-uninfected primary LECs. (C) Green fluorescence phase-contrast images of WT, ΔvIRF3, and R-ΔvIRF3 recombinant KSHV-infected primary LECs in matrigel are shown.
FIG 4
FIG 4
vIRF3-HDAC5 interaction decreases HDAC5 phosphorylation, blocking phosphorylation-dependent HDAC5 translocation. (A) Silver-stained purified V5-labeled vIRF3 complexes at 48 h after Doxy treatment of tetracycline-inducible V5-vIRF3-expressing cell lines. Asterisk, V5-vIRF3; arrow, HDAC5; IP, immunoprecipitation. (B) Interaction of vIRF3-HDAC5. Upon stimulation with Doxy (1 μg/ml), we performed IP with anti-V5, followed by IB with anti-HDAC5. (C) Effect of vIRF3 on the localization of HDAC5. HeLa cells were transiently transfected with the indicated constructs. Cells were then treated with PMA (1 μM) for 2 h before immunofluorescence staining was performed. Arrows indicate the cells coexpressing vIRF3 and HDAC5. (D) Effects of vIRF3 expression on the phosphorylation of HDAC5. At 24 h posttreatment with Doxy (1 μg/ml), TREx/BCBL-1 vector and TREx/BCBL-1 V5/vIRF3 cells were also treated with PMA (1 μM) for 2 h before being harvested. Equal amounts of total proteins were analyzed by IB using anti-HDAC5 pS498-specific antibody. (E) vIRF3 effect on HDAC5 phosphorylation in LECs. LEC-vector and LEC-V5/vIRF3 were harvested and IB was performed with anti-HDAC5 pS498-specific antibody along with other antibodies.
FIG 5
FIG 5
vIRF3 deregulates HDAC5 localization in LECs but not in BECs. Immunofluorescence staining for HDAC5 and vIRF3 was performed. BEC or LEC cells stably expressing vector or V5-vIRF3 were fixed and viewed by confocal microscopy using either anti-HDAC5 (green) or anti-V5 (red) antibodies. Nucleus (blue) was detected with Topro-3 staining.
FIG 6
FIG 6
Alteration of the global gene expression of LECs by vIRF3 and the essential role of HDAC5 for vIRF3-mediated sprouting formation. (A) (Left panel) Principal component plot of both lymphatic endothelium gene expression and blood endothelium gene expression profiles upon vIRF3 expression. Numbers in brackets indicate percentages of total variation explained by each principal component (PC). (Right panel) Venn diagram showing the number of significantly up- and downregulated genes upon vIRF3 expression in LECs and BECs. (B and C) Gene set enrichment analysis (GSEA) of genes significantly up- or downregulated upon vIRF3 induction in LECs (B) and BECs (C), respectively. Positive normalized enrichment scores (NES) indicate increased pathway activity, and negative scores indicate decreased pathway activity. (D and E) The essential role of HDAC5 in vIRF3-induced sprouting formation. Phase-contrast images are shown. (D) LEC-E6/E7 V5/vIRF3 cells were transfected with siRNA as a control (Scramble) or with HDAC5-specific siRNA for 48 h, followed by embedding into matrigel. NC, negative control. (E) Sprouting of LEC-E6/E7 V5/vIRF3 spheroids treated with 5 μM MC1586 (HDAC5 inhibitor).
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