Kaposi's Sarcoma-associated Herpesvirus miRNAs Research Articles

Activation of glucocorticoid receptor signaling inhibits KSHV-induced inflammation and tumorigenesis.

Kaposi's sarcoma (KS) is the most common cancer in HIV-infected patients caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Hyperinflammation is the hallmark of KS. In this study, we have shown that KSHV mediates hyperinflammation by inducing IL-1α and suppressing IL-1Ra. Mechanistically, KSHV miRNAs and vFLIP induce hyperinflammation by activating the NF-κB pathway. A common anti-inflammatory agent dexamethasone blocks KSHV-induced hyperinflammation and tumorigenesis by activating glucocorticoid receptor signaling to suppress IL-1α and induce IL-1Ra. This work has identified IL-1-mediated inflammation as a potential therapeutic target and dexamethasone as a potential therapeutic agent for KSHV-induced malignancies.

Kaposi's Sarcoma-Associated Herpesvirus Latency Locus Renders B Cells Hyperresponsive to Secondary Infections.

Kaposi's sarcoma-associated herpesvirus (KSHV) induces B cell hyperplasia and neoplasia, such as multicentric Castleman's disease (MCD) and primary effusion lymphoma (PEL). To explore KSHV-induced B cell reprogramming in vivo, we expressed the KSHV latency locus, inclusive of all viral microRNAs (miRNAs), in B cells of transgenic mice in the absence of the inhibitory FcγRIIB receptor. The BALB/c strain was chosen as this is the preferred model to study B cell differentiation. The mice developed hyperglobulinemia, plasmacytosis, and B lymphoid hyperplasia. This phenotype was ameliorated by everolimus, which is a rapamycin derivative used for the treatment of mantle cell lymphoma. KSHV latency mice exhibited hyperresponsiveness to the T-dependent (TD) antigen mimic anti-CD40 and increased incidence of pristane-induced inflammation. Lastly, the adaptive immunity against a secondary infection with Zika virus (ZIKV) was markedly enhanced. These phenotypes are consistent with KSHV lowering the activation threshold of latently infected B cells, which may be beneficial in areas of endemicity, where KSHV is acquired in childhood and infections are common.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) establishes latency in B cells and is stringently linked to primary effusion lymphoma (PEL) and the premalignant B cell hyperplasia multicentric Castleman's disease (MCD). To investigate potential genetic background effects, we expressed the KSHV miRNAs in BALB/c transgenic mice. BALB/c mice are the preferred strain for B cell hybridoma development because of their propensity to develop predictable B cell responses to antigen. The BALB/c latency mice exhibited a higher incidence of B cell hyperplasia as well as sustained hyperglobulinemia. The development of neutralizing antibodies against ZIKV was augmented in BALB/c latency mice. Hyperglobulinemia was dampened by everolimus, a derivative of rapamycin, suggesting a role for mTOR inhibitors in managing immune activation, which is hallmark of KSHV infection as well as HIV infection.

Abstract 5140: Lipoxins and their role in Kaposi's sarcoma-associated herpesvirus (KSHV) infection and pathogenesis

Abstract Lipoxins are host anti-inflammatory molecules, which play a vital role in restoring tissue homeostasis. The efficacy of lipoxins and their analog epilipoxins in treating inflammation and related diseases have been well documented. Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) are two well-known inflammation-related diseases caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Controlling inflammation is one of the strategies adopted to treat KS and PEL, a primary motivation for exploring and evaluating the therapeutic potential of using lipoxins. Our study demonstrates how KSHV manipulates and downregulates the secretion of the anti-inflammatory lipoxin A4 in host cells and the viral factors involved in this process using in vitro KS and PEL cells as models. Presence of lipoxin receptor [ALX/Formyl peptidyl receptor (FPR)] in human KS patient tissue sections and in vitro KS and PEL cell models offers a novel possibility for treating KS and PEL with lipoxins. Treating de novo KSHV-infected endothelial cells with lipoxin and epilipoxin creates an anti-inflammatory environment by decreasing levels of NFκB, AKT, ERK1/2, COX-2, and 5-lipoxygenase. Lipoxin treatment on CRISPR/CAS9 technology-mediated ALX/FPR gene-deleted U2OS cells revealed the importance of the lipoxin receptor ALX for effective lipoxin signaling. A viral miRNA cluster was identified as the primary factor contributing to the downregulation of lipoxin A4 secretion in host cells. The KSHV miRNA cluster probably targets enzyme 15-lipoxygenase, which is involved in lipoxin A4 synthesis. Lipoxin treatment in KSHV-infected cells has been shown to reduce cell proliferation and cell survival. Latency factor LANA-1 levels were found to decrease on lipoxin and epilipoxin treatment. Several pro-apoptotic gene levels were upregulated on lipoxin treatment. Several host transcription factors were found to play an important role in regulating KSHV life cycle. This study provides a new insight into the treatment of KS and PEL using nature's own anti-inflammatory molecule, lipoxin. Citation Format: Neelam Sharma-Walia, Jayashree A. Chandrasekharan. Lipoxins and their role in Kaposi's sarcoma-associated herpesvirus (KSHV) infection and pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5140.

Modified Cross-Linking, Ligation, and Sequencing of Hybrids (qCLASH) Identifies Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Targets in Endothelial Cells.

Kaposi's sarcoma (KS) tumors are derived from endothelial cells and express Kaposi's sarcoma-associated herpesvirus (KSHV) microRNAs (miRNAs). Although miRNA targets have been identified in B cell lymphoma-derived cells and epithelial cells, little has been done to characterize the KSHV miRNA targetome in endothelial cells. A recent innovation in the identification of miRNA targetomes, cross-linking, ligation, and sequencing of hybrids (CLASH), unambiguously identifies miRNAs and their targets by ligating the two species while both species are still bound within the RNA-induced silencing complex (RISC). We developed a streamlined quick CLASH (qCLASH) protocol that requires a lower cell input than the original method and therefore has the potential to be used on patient biopsy samples. Additionally, we developed a fast-growing, KSHV-negative endothelial cell line derived from telomerase-immortalized vein endothelial long-term culture (TIVE-LTC) cells. qCLASH was performed on uninfected cells and cells infected with either wild-type KSHV or a mutant virus lacking miR-K12-11/11*. More than 1,400 cellular targets of KSHV miRNAs were identified. Many of the targets identified by qCLASH lacked a canonical seed sequence match. Additionally, most target regions in mRNAs originated from the coding DNA sequence (CDS) rather than the 3' untranslated region (UTR). This set of genes includes some that were previously identified in B cells and some new genes that warrant further study. Pathway analysis of endothelial cell targets showed enrichment in cell cycle control, apoptosis, and glycolysis pathways, among others. Characterization of these new targets and the functional consequences of their repression will be important in furthering our understanding of the role of KSHV miRNAs in oncogenesis.IMPORTANCE KS lesions consist of endothelial cells latently infected with KSHV. Cells that make up these lesions express KSHV miRNAs. Identification of the targets of KSHV miRNAs will help us understand their role in viral oncogenesis. The cross-linking and sequencing of hybrids (CLASH) protocol is a method for unambiguously identifying miRNA targetomes. We developed a streamlined version of CLASH, called quick CLASH (qCLASH). qCLASH requires a lower initial input of cells than for its parent protocol. Additionally, a new fast-growing KSHV-negative endothelial cell line, named TIVE-EX-LTC cells, was established. qCLASH was performed on TIVE-EX-LTC cells latently infected with wild-type (WT) KSHV or a mutant virus lacking miR-K12-11/11*. A number of novel targets of KSHV miRNAs were identified, including targets of miR-K12-11, the ortholog of the cellular oncogenic miRNA (oncomiR) miR-155. Many of the miRNA targets were involved in processes related to oncogenesis, such as glycolysis, apoptosis, and cell cycle control.

Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded microRNAs promote matrix metalloproteinases (MMPs) expression and pro-angiogenic cytokine secretion in endothelial cells.

The human oncogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to Kaposi's sarcoma (KS), a tumor of endothelial cells characterized by angiogenesis and invasiveness. KSHV genome encodes 25 mature microRNAs (miRNAs), but their roles in KSHV-induced tumor dissemination and angiogenesis are not fully understood. In this study, we constructed the sensor reporters of KSHV miRNAs and used a luciferase reporter assay to demonstrate the function of the mimics of KSHV miRNAs. Then, we examined the expression of matrix metalloproteinases (MMPs) and pro-angiogenic cytokines that are related to cell migration and angiogenesis in the KSHV 25 miRNAs transfected endothelial cells. We found that all KSHV miRNAs increased the expression of the transcripts of MMP1, MMP13, VEGFA, and VEGFR2 in different degrees, as well as the secretion of VEGFA protein in the supernatant of endothelial cells. Our results reveal that KSHV miRNAs contribute to regulating MMPs and expression of pro-angiogenic factors, thus, suggesting that these miRNAs might play a crucial role in KSHV-induced cell motility and angiogenesis.

Altering the Anti-inflammatory Lipoxin Microenvironment: a New Insight into Kaposi's Sarcoma-Associated Herpesvirus Pathogenesis.

KSHV infection has been shown to upregulate several host proinflammatory factors, which aid in its survival and pathogenesis. The influence of KSHV infection on anti-inflammatory molecules is not well studied. Since current treatment methods for KS and PEL are fraught with unwanted side effects and low efficiency, the search for new therapeutics is therefore imperative. The use of nature's own molecule lipoxin as a drug is promising. This study opens up new domains in KSHV research focusing on how the virus modulates lipoxin secretion and warrants further investigation of the therapeutic potential of lipoxin using in vitro cell models for KS and PEL.

Kaposi's sarcoma-associated herpesvirus microRNAs repress breakpoint cluster region protein expression, enhance Rac1 activity, and increase in vitro angiogenesis.

MicroRNAs (miRNAs) are small, ∼ 22-nucleotide-long RNAs that regulate gene expression posttranscriptionally. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-miRNAs during latency, and the functional significance of these microRNAs during KSHV infection and their cellular targets have been emerging recently. Using a previously reported microarray profiling analysis, 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 and during KSHV infection. Luciferase assays wherein the Bcr 3' untranslated region (UTR) was cloned downstream of a luciferase reporter showed repression in the presence of miR-K6-5, and mutation of one of the two predicted miR-K6-5 binding sites relieved this repression. Furthermore, inhibition or deletion of miR-K6-5 in KSHV-infected cells showed increased Bcr protein levels. Together, these results show that Bcr is a direct target of the KSHV miRNA miR-K6-5. To understand the functional significance of Bcr knockdown in the context of KSHV-associated disease, we hypothesized that the knockdown of Bcr, a negative regulator of Rac1, might enhance Rac1-mediated angiogenesis. We found that HUVECs transfected with miR-K6-5 had increased Rac1-GTP levels and tube formation compared to HUVECs transfected with control miRNAs. Knockdown of Bcr in latently KSHV-infected BCBL-1 cells increased the levels of viral RTA, suggesting that Bcr repression by KSHV might aid lytic reactivation. Together, our results reveal a new function for both KSHV miRNAs and Bcr in KSHV infection and suggest that KSHV miRNAs, in part, promote angiogenesis and lytic reactivation. Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) infection is linked to multiple human cancers and lymphomas. KSHV encodes small nucleic acids (microRNAs) that can repress the expression of specific human genes, the biological functions of which are still emerging. This report uses a variety of approaches to show that a KSHV microRNA represses the expression of the human gene called breakpoint cluster region (Bcr). Repression of Bcr correlated with the activation of a protein previously shown to cause KS-like lesions in mice (Rac1), an increase in KS-associated phenotypes (tube formation in endothelial cells and vascular endothelial growth factor [VEGF] synthesis), and modification of the life cycle of the virus (lytic replication). Our results suggest that KSHV microRNAs suppress host proteins and contribute to KS-associated pathogenesis.

KSHV miRNAs Decrease Expression of Lytic Genes in Latently Infected PEL and Endothelial Cells by Targeting Host Transcription Factors

Kaposi’s sarcoma-associated herpesvirus (KSHV) microRNAs are encoded in the latency-associated region. Knockdown of KSHV miR-K12-3 and miR-K12-11 increased expression of lytic genes in BC-3 cells, and increased virus production from latently infected BCBL-1 cells. Furthermore, iSLK cells infected with miR-K12-3 and miR-K12-11 deletion mutant viruses displayed increased spontaneous reactivation and were more sensitive to inducers of reactivation than cells infected with wild type KSHV. Predicted binding sites for miR-K12-3 and miR-K12-11 were found in the 3’UTRs of the cellular transcription factors MYB, Ets-1, and C/EBPα, which activate RTA, the KSHV replication and transcription activator. Targeting of MYB by miR-K12-11 was confirmed by cloning the MYB 3’UTR downstream from the luciferase reporter. Knockdown of miR‑K12-11 resulted in increased levels of MYB transcript, and knockdown of miR-K12-3 increased both C/EBPα and Ets-1 transcripts. Thus, miR-K12-11 and miR-K12-3 contribute to maintenance of latency by decreasing RTA expression indirectly, presumably via down‑regulation of MYB, C/EBPα and Ets-1, and possibly other host transcription factors.

Genomewide Mapping and Screening of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) 3′ Untranslated Regions Identify Bicistronic and Polycistronic Viral Transcripts as Frequent Targets of KSHV MicroRNAs

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes over 90 genes and 25 microRNAs (miRNAs). The KSHV life cycle is tightly regulated to ensure persistent infection in the host. In particular, miRNAs, which primarily exert their effects by binding to the 3' untranslated regions (3'UTRs) of target transcripts, have recently emerged as key regulators of KSHV life cycle. Although studies with RNA cross-linking immunoprecipitation approach have identified numerous targets of KSHV miRNAs, few of these targets are of viral origin because most KSHV 3'UTRs have not been characterized. Thus, the extents of viral genes targeted by KSHV miRNAs remain elusive. Here, we report the mapping of the 3'UTRs of 74 KSHV genes and the effects of KSHV miRNAs on the control of these 3'UTR-mediated gene expressions. This analysis reveals new bicistronic and polycistronic transcripts of KSHV genes. Due to the 5'-distal open reading frames (ORFs), KSHV bicistronic or polycistronic transcripts have significantly longer 3'UTRs than do KSHV monocistronic transcripts. Furthermore, screening of the 3'UTR reporters has identified 28 potential new targets of KSHV miRNAs, of which 11 (39%) are bicistronic or polycistronic transcripts. Reporter mutagenesis demonstrates that miR-K3 specifically targets ORF31-33 transcripts at the lytic locus via two binding sites in the ORF33 coding region, whereas miR-K10a-3p and miR-K10b-3p and their variants target ORF71-73 transcripts at the latent locus through distinct binding sites in both 5'-distal ORFs and intergenic regions. Our results indicate that KSHV miRNAs frequently target the 5'-distal coding regions of bicistronic or polycistronic transcripts and highlight the unique features of KSHV miRNAs in regulating gene expression and life cycle.

Proteomic screening of human targets of viral microRNAs reveals functions associated with immune evasion and angiogenesis.

Kaposi's sarcoma (KS) is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV). The virus expresses unique microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC) in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1) following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α), and up-regulation of a protein involved in stimulating angiogenesis (HMOX1). This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.

Viral Ortholog Complements Mir-155 Deficiency-Associated Abnormalities and Together with Additional Viral Mirnas Causes B Cell Hyperplasia and B Cell Lymphoma

AbstractAbstract 4866MicroRNA-155 (miR-155) is overexpressed in many types of cancers including B cell lymphomas. It has also an essential function in GC development. These functions are conserved among species and miR-155 knockout (ko) mice are deficient in GC development in Peyer’s patches (PP) and lymph nodes. The human tumor virus Kaposi sarcoma associated herpesvirus (KSHV) encodes an ortholog to miR-155, named miR-K12-11. MiR-155 and miR-K12-11 share 100% seed sequence homology (Nature. 2007 Dec 13;450(7172):1096-9, J Virol. 2007 Dec;81(23):12836-45). Another human herpesvirus, Epstein-Barr Virus, induces mir-155 upon infection and immortalization of naïve B cells in culture. To test the hypothesis that miR-K12-11 can complement the normal function of miR-155 in vivo, we generated transgenic mice, which express miR-K12-11, as well as the other 21 KSHV miRNAs. Using this mouse model, we show that KSHV latency locus which contains a viral ortholog of miR-155, KSHV miR-K12-11 complements B cell abnormalities associated with the lack of miR-155. Germinal center (GC) formation is rescued in spleen and PP and lowered immunoglobulin level is also augmented to normal in KSHV latency locus transgenic mice with miR-155 ko background. Furthermore, mature B cells were chronically activated, leading to hyperglobulinemia triggered by increased plasma cell frequency. Marginal zone (MZ) B cells developed hyperplasia and the mice had an augmented response to T-dependent antigen as well as the TLR4 ligand LPS in vivo. This suggests that by mimicking miR-155 (and other functions) this human tumor virus drives the expansion of infected B cells, which if left unchecked can accumulate secondary mutations leading to post-GC lymphoma and multicentric Castleman’s disease. Disclosures:No relevant conflicts of interest to declare.

Ago HITS-CLIP Expands Understanding of Kaposi's Sarcoma-associated Herpesvirus miRNA Function in Primary Effusion Lymphomas

KSHV is the etiological agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentricCastleman's disease (MCD). The fact that KSHV-encoded miRNAs are readily detectable in all KSHV-associated tumors suggests a potential role in viral pathogenesis and tumorigenesis. MiRNA-mediated regulation of gene expression is a complex network with each miRNA having many potential targets, and to date only few KSHV miRNA targets have been experimentally determined. A detailed understanding of KSHV miRNA functions requires high-through putribonomics to globally analyze putative miRNA targets in a cell type-specific manner. We performed Ago HITS-CLIP to identify viral and cellular miRNAs and their cognate targets in two latently KSHV-infected PEL cell lines. Ago HITS-CLIP recovered 1170 and 950 cellular KSHVmiRNA targets from BCBL-1 and BC-3, respectively. Importantly, enriched clusters contained KSHV miRNA seed matches in the 3′UTRs of numerous well characterized targets, among them THBS1, BACH1, and C/EBPβ. KSHV miRNA targets were strongly enriched for genes involved in multiple pathways central for KSHV biology, such as apoptosis, cell cycle regulation, lymphocyte proliferation, and immune evasion, thus further supporting a role in KSHV pathogenesis and potentially tumorigenesis. A limited number of viral transcripts were also enriched by HITS-CLIP including vIL-6 expressed only in a subset of PEL cells during latency. Interestingly, Ago HITS-CLIP revealed extremely high levels of Ago-associated KSHV miRNAs especially in BC-3 cells where more than 70% of all miRNAs are of viral origin. This suggests that in addition to seed match-specific targeting of cellular genes, KSHV miRNAs may also function by hijacking RISCs, thereby contributing to a global de-repression of cellular gene expression due to the loss of regulation by human miRNAs. In summary, we provide an extensive list of cellular and viral miRNA targets representing an important resource to decipher KSHV miRNA function.

Genetic variation in KSHV encoded microRNAs affects microRNA expression and is associated with multicentric Castleman’s disease risk

Kaposi sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs which potentially yield 25 mature microRNAs and have been shown to play prominent roles in the viral lifecycle including maintaining viral latency, evading the host immune response, and controlling lytic replication. We previously reported phylogenetic analysis of the microRNA-coding region of KSHV from Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD) patients. We showed a high level of conservation for most sequences, but also a divergent cluster of 5 KSHV sequences including 2 from MCD patients [1]. We additionally observed single nucleotide polymorphisms (SNP) in the sequence of KSHV encoded mature and pre-miRNAs from clinical samples including a SNP in mir-K12-5 reported to result in increased expression of the mature miRNA [2]. To determine whether SNPs in other KSHV encoded miRNAs resulted in differences in miRNA processing and expression we used four complimentary approaches. Analysis of KSHV miRNA expression levels in PEL cell lines using custom ABI real time qPCR assays showed differential expression that correlates with sequence. Lentiviral vectors constructed to express wild type and variant pre-miRNAs were transduced into 293T cells to make stably expressing cell lines. miRNA expression was assessed using custom ABI real time qPCR assays. Luciferase reporter assays were performed following transient transfections of each miRNA. In addition, in vitro maturation assays were performed to assess differences in Drosha/DGCR8 and Dicer cleavage between wild type and variant pre-miRNAs. Our results indicate that polymorphisms within the pre-miRNA sequence can cause subtle expression differences as in the case of KSHV miR-K12-6 or more profound changes as observed in miR-K12-5. Our data clearly shows that SNPs can affect pre-miRNA processing resulting in changes in mature miRNA expression levels. To extend our studies on miRNA variation in MCD patients, KSHV miRNA sequences from 23 MCD patients and 7 patients with a newly described KSHV-associated inflammatory cytokine syndrome (KICS) were examined by amplification, cloning, and sequencing of a 646-bp fragment of K12/T0.7 encoding miRNA-K12-10 and miRNA-K12-12 and a 2.8-kbp fragment containing the remaining 10 pre-microRNAs. Phylogenetic analysis showed a distinct variant cluster consisting exclusively of MCD and KICS patients in all trees. Pearson’s chi-squared analysis revealed 40 single nucleotide polymorphisms (SNPs) at various loci were significantly associated with MCD and KICS risk. Additionally, cluster analysis of these SNPs generated several combinations of three SNPs as putative indicators of MCD and KICS risk. Taken together, these findings show that MCD and KICS patients frequently have unusual KSHV microRNA sequences and suggest association between the observed sequence variation and risk of MCD and KICS.

KSHV-Encoded MicroRNAs: Lessons for Viral Cancer Pathogenesis and Emerging Concepts.

The human genome contains microRNAs (miRNAs), small noncoding RNAs that orchestrate a number of physiologic processes through regulation of gene expression. Burgeoning evidence suggests that dysregulation of miRNAs may promote disease progression and cancer pathogenesis. Virus-encoded miRNAs, exhibiting unique molecular signatures and functions, have been increasingly recognized as contributors to viral cancer pathogenesis. A large segment of the existing knowledge in this area has been generated through characterization of miRNAs encoded by the human gamma-herpesviruses, including the Kaposi's sarcoma-associated herpesvirus (KSHV). Recent studies focusing on KSHV miRNAs have led to a better understanding of viral miRNA expression in human tumors, the identification of novel pathologic check points regulated by viral miRNAs, and new insights for viral miRNA interactions with cellular (“human”) miRNAs. Elucidating the functional effects of inhibiting KSHV miRNAs has also provided a foundation for further translational efforts and consideration of clinical applications. This paper summarizes recent literature outlining mechanisms for KSHV miRNA regulation of cellular function and cancer-associated pathogenesis, as well as implications for interactions between KSHV and human miRNAs that may facilitate cancer progression. Finally, insights are offered for the clinical feasibility of targeting miRNAs as a therapeutic approach for viral cancers.

Viral MicroRNA Targetome of KSHV-Infected Primary Effusion Lymphoma Cell Lines

Primary effusion lymphoma (PEL) is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) and frequently also harbors Epstein-Barr virus (EBV). The expression of KSHV- and EBV-encoded microRNAs (miRNAs) in PELs suggests a role for these miRNAs in latency and lymphomagenesis. Using PAR-CLIP, a technology which allows the direct and transcriptome-wide identification of miRNA targets, we delineate the target sites for all viral and cellular miRNAs expressed in PEL cell lines. The resulting data set revealed that KSHV miRNAs directly target more than 2000 cellular mRNAs, including many involved in pathways relevant to KSHV pathogenesis. Moreover, 58% of these mRNAs are also targeted by EBV miRNAs, via distinct binding sites. In addition to a known viral analog of cellular miR-155, we show that KSHV encodes a viral miRNA that mimics cellular miR-142-3p function. In summary, this study identifies an extensive list of KSHV miRNA targets, which are likely to influence viral replication and pathogenesis.

MicroRNAs encoded by Kaposi's sarcoma‐associated herpesvirus regulate viral life cycle

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked with Kaposi's sarcoma and lymphomas. The pathogenesis of KSHV depends on the balance between two phases of the viral cycle: latency and lytic replication. In this study, we report that KSHV-encoded microRNAs (miRNAs) function as regulators by maintaining viral latency and inhibiting viral lytic replication. MiRNAs are short, noncoding, small RNAs that post-transcriptionally regulate the expression of messenger RNAs. Of the 12 viral miRNAs expressed in latent KSHV-infected cells, we observed that expression of miR-K3 can suppress both viral lytic replication and gene expression. Further experiments indicate that miR-K3 can regulate viral latency by targeting nuclear factor I/B. Nuclear factor I/B can activate the promoter of the viral immediate-early transactivator replication and transcription activator (RTA), and depletion of nuclear factor I/B by short hairpin RNAs had similar effects on the viral life cycle to those of miR-K3. Our results suggest a role for KSHV miRNAs in regulating the viral life cycle.

Analysis of rhesus rhadinovirus microRNAs expressed in virus-induced tumors from infected rhesus macaques

Rhesus rhadinovirus (RRV), a primate γ-herpesvirus related to human Kaposi's sarcoma-associated herpesvirus (KSHV), causes a similar pattern of pathogenesis. Previously, RRV was shown to express 7 pre-microRNAs (pre-miRNAs) in latently infected cells. Using deep sequencing, we analyzed the pattern of small RNA expression in vivo using latently RRV-infected B-cell lymphoma and retroperitoneal fibromatosis tissues. We identified 15 virally encoded pre-miRNAs in both tumors, including all previously reported RRV pre-miRNAs. Although all 15 RRV pre-miRNAs, like all 12 KSHV pre-miRNAs, are located 3′ to the conserved viral ORF71 gene and in the same transcriptional orientation, only one RRV miRNA is homologous to a KSHV miRNA. One previously identified RRV miRNA, miR-rR1-3, is actually a miRNA offset RNA (moRNA) derived from sequences located adjacent to pre-miR-rR1-3. Several other RRV-derived moRNAs were obtained, including one recovered >600 times. Together, this research provides a comprehensive list of the miRNAs and moRNAs encoded by RRV.

Small RNA profiling reveals antisense transcription throughout the KSHV genome and novel small RNAs

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus that encodes 12 precursor microRNAs (pre-miRNAs) that give rise to 17 different known approximately 22-nucleotide (nt) effector miRNAs. Like all herpesviruses, KSHV has two modes of infection: (1) a latent mode whereby only a subset of viral genes are expressed and (2) a lytic mode during which the full remaining viral genes are expressed. To date, KSHV miRNAs have been mostly identified via analysis of cells that are undergoing latent infection. Here, we developed a method to profile small RNAs ( approximately 18-75 nt) from populations of cells undergoing predominantly lytic infection. Using two different next-generation sequencing platforms, we cloned and sequenced both pre-miRNAs and derivative miRNAs. Our analysis shows that the vast majority of viral and host 5p miRNAs are co-terminal with the 5' end of the cloned pre-miRNAs, consistent with both being defined by microprocessor cleavage. We report the complete repertoire (25 total) of 5p and 3p derivative miRNAs from all 12 previously described KSHV pre-miRNAs. Two KSHV pre-miRNAs, pre-miR-K12-8 and pre-miR-K12-12, encode abundant derivative miRNAs from the previously unreported strands of the pre-miRNA. We identify several novel small RNAs of low abundance, including viral miRNA-offset-RNAs (moRNAs), and antisense viral miRNAs (miRNA-AS) that are encoded antisense to previously reported KSHV pre-miRNAs. Finally, we observe widespread antisense transcription relative to known coding sequences during lytic replication. Despite the enormous potential to form double-stranded RNA in KSHV-infected cells, we observe no evidence for the existence of abundant viral-derived small interfering RNAs (siRNAs).

Systematic Analysis of Viral and Cellular MicroRNA Targets in Cells Latently Infected with Human γ-Herpesviruses by RISC Immunoprecipitation Assay

The mRNA targets of microRNAs (miRNAs) can be identified by immunoprecipitation of Argonaute (Ago) protein-containing RNA-induced silencing complexes (RISCs) followed by microarray analysis (RIP-Chip). Here we used Ago2-based RIP-Chip to identify transcripts targeted by Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs (n = 114), Epstein-Barr virus (EBV) miRNAs (n = 44), and cellular miRNAs (n = 2337) in six latently infected or stably transduced human B cell lines. Of the six KSHV miRNA targets chosen for validation, four showed regulation via their 3'UTR, while two showed regulation via binding sites within coding sequences. Two genes governing cellular transport processes (TOMM22 and IPO7) were confirmed to be targeted by EBV miRNAs. A significant number of viral miRNA targets were upregulated in infected cells, suggesting that viral miRNAs preferentially target cellular genes induced upon infection. Transcript half-life both of cellular and viral miRNA targets negatively correlated with recruitment to RISC complexes, indicating that RIP-Chip offers a quantitative estimate of miRNA function.

A Human Herpesvirus MicroRNA Inhibits p21 Expression and Attenuates p21-Mediated Cell Cycle Arrest

The oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) expresses 12 viral microRNAs (miRNAs) in latently infected cells. Here, we report that cellular mRNAs encoding the cellular cyclin-dependent kinase inhibitor p21, a key inducer of cell cycle arrest, are direct targets for KSHV miR-K1. Ectopically expressed KSHV miR-K1 specifically inhibited the expression of endogenous p21 in KSHV-negative cells and strongly attenuated the cell cycle arrest that normally occurs upon p53 activation, yet miR-K1 did not prevent the induction of other p53-responsive genes. Stable knockdown of miR-K1 in latently KSHV-infected human primary effusion lymphoma (PEL) B cells revealed a derepression of p21 expression and enhanced cell cycle arrest following activation of p53. Our data demonstrate that miR-K1 represses the expression of p21, a protein with known tumor suppressor functions, and suggest that this KSHV miRNA is likely to contribute to the oncogenic potential of this opportunistic viral pathogen.