Kaposi’s sarcoma-associated herpesvirus (KSHV) is causally linked to several acquired immune deficiency syndrome related malignancies including Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentric Castleman’s disease1. Control of viral lytic replication is essential for KSHV latency, evasion of host immune system, and induction of tumors1. Here, we show that deletion of a cluster of 14 microRNAs (miRs) from KSHV genome significantly enhances viral lytic replication as a result of reduced NF-κB activity. The miR cluster regulates NF-κB pathway by reducing the expression of IκBα protein, an inhibitor of the NF-κB complexes. Computational and miR seed mutagenesis analyses identify KSHV miR-K1 that directly mediates IκBα ?protein level by targeting the 3’UTR of its transcript. Expression of miR-K1 is sufficient to rescue the NF-κB activity and inhibit viral lytic replication while inhibition of miR-K1 in KSHV-infected PEL cells has the opposite effects. Thus, KSHV encodes a miR to control viral replication by activating NF-κB pathway. These results illustrate an important role for KSHV miRs in regulating viral latency and lytic replication by manipulating a host survival pathway.

SUMMARY Brown adipose tissue (BAT) protects against obesity by promoting energy expenditure via uncoupled respiration. To uncover BAT-specific long non-coding RNAs (lncRNAs), we used RNA-seq to reconstruct de novo transcriptomes of mouse brown, inguinal white, and epididymal white fat and identified ~1500 lncRNAs, including 127 BAT-restricted loci induced during differentiation and often targeted by key regulators PPARγ, C/EBPα and C/EBPβ. One of them, lnc-BATE1, is required for establishment and maintenance of BAT identity and thermogenic capacity. lnc-BATE1 inhibition impairs concurrent activation of brown fat and repression of white fat genes, and is partially rescued by exogenous lnc-BATE1 with mutated siRNA-targeting sites, demonstrating a function in trans. We show that lnc-BATE1 binds heterogeneous nuclear ribonucleoprotein U and that both are required for brown adipogenesis. Our work provides an annotated catalog for the study of fat depot-selective lncRNAs, available online, and establishes lnc-BATE1 as a novel regulator of BAT development and physiology.

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several human cancers, including Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease, malignancies commonly found in HIV-infected patients. While KSHV encodes diverse functional products, its mechanism of oncogenesis remains unknown. In this study, we determined the roles KSHV microRNAs (miRs) in cellular transformation and tumorigenesis using a recently developed KSHV-induced cellular transformation system of primary rat mesenchymal precursor cells. A mutant with a cluster of 10 precursor miRs (pre-miRs) deleted failed to transform primary cells, and instead, caused cell cycle arrest and apoptosis. Remarkably, the oncogenicity of the mutant virus was fully restored by genetic complementation with the miR cluster or several individual pre-miRs, which rescued cell cycle progression and inhibited apoptosis in part by redundantly targeting IκBα and the NF-κB pathway. Genomic analysis identified common targets of KSHV miRs in diverse pathways with several cancer-related pathways preferentially targeted. These works define for the first time an essential viral determinant for KSHV-induced oncogenesis and identify NF-κB as a critical pathway targeted by the viral miRs. Our results illustrate a common theme of shared functions with hierarchical order among the KSHV miRs.

Enhancing brown fat activity and promoting white fat browning are attractive therapeutic strategies for treating obesity and associated metabolic disorders. To provide a comprehensive picture of the gene regulatory network in these processes, we conducted a series of transcriptome studies by RNA sequencing (RNA-seq) and quantified the mRNA and long noncoding RNA (lncRNA) changes during white fat browning (chronic cold exposure, beta-adrenergic agonist treatment, and intense exercise) and brown fat activation or inactivation (acute cold exposure or thermoneutrality, respectively). mRNA–lncRNA coexpression networks revealed dynamically regulated lncRNAs to be largely embedded in nutrient and energy metabolism pathways. We identified a brown adipose tissue–enriched lncRNA, lncBATE10, that was governed by the cAMP-cAMP response element-binding protein (Creb) axis and required for a full brown fat differentiation and white fat browning program. Mechanistically, lncBATE10 can decoy Celf1 from Pgc1α, thereby protecting Pgc1α mRNA from repression by Celf1. Together, these studies provide a comprehensive data framework to interrogate the transcriptomic changes accompanying energy homeostasis transition in adipose tissue.

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is etiologically associated with KS, the most common AIDS-related malignancy. KS is characterized by vast angiogenesis and hyperproliferative spindle cells. We have previously reported that HIV-1 Tat can trigger KSHV reactivation and accelerate Kaposin A-induced tumorigenesis. Here, we explored Tat promotion of KSHV vIL-6-induced angiogenesis and tumorigenesis. Tat promotes vIL-6-induced cell proliferation, cellular transformation, vascular tube formation and VEGF production in culture. Tat enhances vIL-6-induced angiogenesis and tumorigenesis of fibroblasts and human endothelial cells in a chicken chorioallantoic membrane (CAM) model. In an allograft model, Tat promotes vIL-6-induced tumorigenesis and expression of CD31, CD34, SMA, VEGF, b-FGF, and cyclin D1. Mechanistic studies indicated Tat activates PI3K and AKT, and inactivates PTEN and GSK-3β in vIL-6 expressing cells. LY294002, a specific inhibitor of PI3K, effectively impaired Tat’s promotion of vIL-6-induced tumorigenesis. Together, these results provide the first evidence that Tat might contribute to KS pathogenesis by synergizing with vIL-6, and identify PI3K/AKT pathway as a potential therapeutic target in AIDS-related KS patients.

Kaposi's sarcoma (KS) is a highly disseminated angiogenic tumor of endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced tumor dissemination and metastasis remain unknown. Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion. Bioinformatics and luciferase reporter analyses showed that miR-K3 directly targeted G protein-coupled receptor (GPCR) kinase 2 (GRK2, official gene symbol ADRBK1). Importantly, overexpression of GRK2 reversed miR-K3 induction of cell migration and invasion. Furthermore, the chemokine receptor CXCR2, which was negatively regulated by GRK2, was upregulated in miR-K3-transduced endothelial cells. Knock down of CXCR2 abolished miR-K3-induced cell migration and invasion. Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT. Both CXCR2 induction and the release of AKT from GRK2 were required for miR-K3 maximum activation of AKT and induction of cell migration and invasion. Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion. Our data provide the first-line evidence that, by repressing GRK2, miR-K3 facilitates cell migration and invasion via activation of CXCR2/AKT signaling, which likely contribute to the dissemination of KSHV-induced tumors.

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