kaposi's sarcoma-associated herpesvirus micrornas repress

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  • Kaposis Sarcoma-Associated Herpesvirus MicroRNAs RepressBreakpoint Cluster Region Protein Expression, Enhance Rac1 Activity,and Increase In Vitro Angiogenesis

    Dhivya Ramalingam, Christine Happel, Joseph M. Ziegelbauer

    HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

    ABSTRACT

    MicroRNAs (miRNAs) are small, 22-nucleotide-long RNAs that regulate gene expression posttranscriptionally. Kaposis sarco-ma-associated herpesvirus (KSHV) encodes 12 pre-miRNAs during latency, and the functional significance of these microRNAsduring KSHV infection and their cellular targets have been emerging recently. Using a previously reported microarray profilinganalysis, we identified breakpoint cluster region mRNA (Bcr) as a cellular target of the KSHV miRNA miR-K12-6-5p (miR-K6-5).Bcr protein levels were repressed in human umbilical vein endothelial cells (HUVECs) upon transfection with miR-K6-5 andduring KSHV infection. Luciferase assays wherein the Bcr 3= untranslated region (UTR) was cloned downstream of a luciferasereporter showed repression in the presence of miR-K6-5, and mutation of one of the two predicted miR-K6-5 binding sites re-lieved this repression. Furthermore, inhibition or deletion of miR-K6-5 in KSHV-infected cells showed increased Bcr proteinlevels. Together, these results show that Bcr is a direct target of the KSHV miRNA miR-K6-5. To understand the functional sig-nificance of Bcr knockdown in the context of KSHV-associated disease, we hypothesized that the knockdown of Bcr, a negativeregulator of Rac1, might enhance Rac1-mediated angiogenesis. We found that HUVECs transfected with miR-K6-5 had in-creased Rac1-GTP levels and tube formation compared to HUVECs transfected with control miRNAs. Knockdown of Bcr in la-tently KSHV-infected BCBL-1 cells increased the levels of viral RTA, suggesting that Bcr repression by KSHV might aid lytic re-activation. Together, our results reveal a new function for both KSHV miRNAs and Bcr in KSHV infection and suggest thatKSHV miRNAs, in part, promote angiogenesis and lytic reactivation.

    IMPORTANCE

    Kaposis sarcoma (KS)-associated herpesvirus (KSHV) infection is linked to multiple human cancers and lymphomas. KSHVencodes small nucleic acids (microRNAs) that can repress the expression of specific human genes, the biological functions ofwhich are still emerging. This report uses a variety of approaches to show that a KSHV microRNA represses the expression of thehuman gene called breakpoint cluster region (Bcr). Repression of Bcr correlated with the activation of a protein previouslyshown to cause KS-like lesions in mice (Rac1), an increase in KS-associated phenotypes (tube formation in endothelial cells andvascular endothelial growth factor [VEGF] synthesis), and modification of the life cycle of the virus (lytic replication). Our re-sults suggest that KSHV microRNAs suppress host proteins and contribute to KS-associated pathogenesis.

    Kaposis sarcoma (KS)-associated herpesvirus (KSHV) is a gam-maherpesvirus that is associated with AIDS-associated KS, pri-mary effusion lymphoma (PEL), and multicentric Castlemans dis-ease (MCD) (1, 2). KSHV infects primarily cells of endothelial cell orB-cell origin and persists in either a latent phase, during which only afew viral genes are expressed, or a lytic phase, where the full repertoireof viral genes is expressed and infectious virions are released. Duringlatent infection, KSHV also expresses 12 pre-microRNAs (pre-miRNAs) that are processed to yield 20 mature miRNAs (36).miRNAs are 22-nucleotide-long RNAs that typically bind with im-perfect complementarity to the 3= untranslated regions (UTRs) ofmRNAs and cause translational repression and mRNA degradation(7). The KSHV miRNAs are believed to be involved in repressingnumerous targets that are involved in immune evasion (MICB) (8),apoptosis (BCLAF1, TWEAKR, and caspase 3) (911), lytic reactiva-tion (RTA) (12, 13), angiogenesis (THBS1) (14), transcription re-pression (BACH1) (15, 16), and cell signaling (p21, IB, andSMAD5) (1719).

    Previously, we reported a microarray-based expression profil-ing approach to identify cellular mRNAs that are downregulatedin the presence of KSHV miRNAs (11). From this array, we iden-

    tified BCLAF1 (11), TWEAKR (9), and IRAK1 and MyD88 (20) ascellular targets of KSHV miRNAs. In this report, we identify thebreakpoint cluster region (Bcr) mRNA and RacGAP1 as cellulartargets of the KSHV miRNA miR-K12-6-5p (miR-K6-5).

    Bcr was originally identified as a fusion partner of Bcr-Abl,which is the fusion protein that is associated with most forms ofchronic myelogenous leukemia (CML) and acute lymphocyticleukemias (ALLs) (21). Bcr by itself has been suggested to act as atumor suppressor (22). Bcr interferes with the -cateninTcf4

    Received 23 December 2014 Accepted 22 January 2015

    Accepted manuscript posted online 28 January 2015

    Citation Ramalingam D, Happel C, Ziegelbauer JM. 2015. Kaposis sarcoma-associated herpesvirus microRNAs repress breakpoint cluster region proteinexpression, enhance Rac1 activity, and increase in vitro angiogenesis. J Virol89:4249 4261. doi:10.1128/JVI.03687-14.

    Editor: R. M. Longnecker

    Address correspondence to Joseph M. Ziegelbauer, ziegelbauerjm@mail.nih.gov.

    Copyright 2015, American Society for Microbiology. All Rights Reserved.

    doi:10.1128/JVI.03687-14

    April 2015 Volume 89 Number 8 jvi.asm.org 4249Journal of Virology

    on February 14, 2018 by guest

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  • interaction and is a negative regulator of the Wnt pathway (22,23). Bcr phosphorylates the Ras effector protein AF-6 and facili-tates its interaction with Ras, thereby inhibiting extracellular sig-nal-regulated kinase (ERK) activation and cellular proliferation(24). The Bcr protein has oligomerization, Ser/Thr kinase (25),and guanosine nucleotide exchange factor (GEF) (26, 27) do-mains. In addition, Bcr contains a C-terminal GTPase activationdomain (GAP), with which it inhibits the function of Rac1 (28).Rac1 exists between an active, membrane-bound state (Rac1-GTP) and an inactive, cytoplasmic state (Rac1-GDP) (29). As aRac1 GAP, Bcr enhances the intrinsic GTPase activity of Rac1 andtherefore negatively regulates its function. Rac1 belongs to theRho family of small GTPases that control cytoskeletal organiza-tion, cell motility, and angiogenesis. Deregulated angiogenesis isoften observed in many cancers and is a hallmark of KS. KS is ahighly vascularized tumor that expresses elevated levels of angio-genic molecules such as basic fibroblast growth factor (bFGF),platelet-derived growth factor (PDGF), and vascular endothelialgrowth factor (VEGF) (30). VEGF is a key proangiogenic factorand, upon binding to its receptors on the endothelial cell surface,stimulates angiogenesis by recruiting Rac1. Small interfering RNA(siRNA)-mediated knockdown of Rac1 reduces VEGF-inducedangiogenesis in vitro and tumor progression in mice (31). Aber-rant regulation of Rac1 has been observed for many cancers, in-cluding KS (3234).

    In this study, we identified Bcr, a negative regulator of Rac1, inthe analysis of mRNAs that were downregulated by KSHVmiRNAs. Bcr was repressed by miR-K6-5 in B cells and endothe-lial cells and in the context of de novo KSHV infection. The repres-sion of Bcr by miR-K6-5 caused increases in the levels of Rac1-GTP and VEGF in endothelial cells. Furthermore, the increasedRac1-GTP levels also enhanced tube length in endothelial tubeformation assays, and this effect was recapitulated by an siRNAtargeting Bcr. Finally, siRNA-mediated knockdown of Bcr in la-tently infected BCBL-1 cells increased the levels of the viral RTAprotein, the key regulator of KSHV lytic reactivation, suggesting apotential advantage of Bcr suppression during KSHV infection.These results together identify a cellular mRNA, Bcr, as a target ofthe KSHV miRNA miR-K6-5, and we further demonstrate thatthe virus is able to establish an angiogenic environment and facil-itate lytic reactivation as a consequence of this repression.

    MATERIALS AND METHODSCell culture and reagents. Human umbilical vein endothelial cells(HUVECs; Lonza) were maintained for up to five passages with completeEGM-2 BulletKit (Lonza). The latently KSHV-infected body cavity-basedlymphoma cell line (BCBL-1) and the uninfected BJAB B-cell line weremaintained in RPMI 1640 supplemented with 10% fetal bovine serum(FBS), 1 penicillin-streptomycin, and 55 M -mercaptoethanol. 293cells and SLK cells (35, 36) were maintained in Dulbeccos modified Ea-gles medium (DMEM) supplemented with 10% FBS and 1 penicillin-streptomycin. KSHV-infected SLK cells (SLKK cells) (37) were main-tained in DMEM supplemented with 10% FBS, penicillin-streptomycin,and 10 g/ml of puromycin. Telomerase-immortalized microvascular en-dothelial (TIME) cells were grown in vascular cell basal medium withendothelial cell growth kit-VEGF (ATCC), and KSHV-infected TIMEcells were grown in medium supplemented with 50 g/ml of hygromycinB. Inducible TIME (iTIME) cells were a gift from Craig McCormick (Dal-housie University) and were generated by using the Retro-X Tet-Off Ad-vanced inducible expression system (Clontech) (38). Clonal TIME celllines expressing Tet-Off Advanced transactivator (tTA-Advanced cells)

    were transduced with retroviral vectors encoding KSHV RTA controlledby a modified Tet-responsive element (TREmod) joined to a minimalcytomegalovirus (CMV) promoter. The clonal cells resulting from thesetransductions, dubbed iTIME cells, were cultured in complete vascularcell basal medium (VCBM) supplemented with a VEGF kit (ATCC) con-taining 500 g/ml G418, 1 g/ml puromycin, and 200 ng/ml doxycycline.KSHV-infected cells were g