microRNA dependent and independent deregulation of long non-coding RNAs by an oncogenic herpesvirus.

Kaposi's sarcoma (KS) is a highly prevalent cancer in AIDS patients, especially in sub-Saharan Africa. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of KS and other cancers like Primary Effusion Lymphoma (PEL). In KS and PEL, all tumors harbor latent KSHV episomes and express latency-associated viral proteins and microRNAs (miRNAs). The exact molecular mechanisms by which latent KSHV drives tumorigenesis are not completely understood. Recent developments have highlighted the importance of aberrant long non-coding RNA (lncRNA) expression in cancer. Deregulation of lncRNAs by miRNAs is a newly described phenomenon. We hypothesized that KSHV-encoded miRNAs deregulate human lncRNAs to drive tumorigenesis. We performed lncRNA expression profiling of endothelial cells infected with wt and miRNA-deleted KSHV and identified 126 lncRNAs as putative viral miRNA targets. Here we show that KSHV deregulates host lncRNAs in both a miRNA-dependent fashion by direct interaction and in a miRNA-independent fashion through latency-associated proteins. Several lncRNAs that were previously implicated in cancer, including MEG3, ANRIL and UCA1, are deregulated by KSHV. Our results also demonstrate that KSHV-mediated UCA1 deregulation contributes to increased proliferation and migration of endothelial cells.

Epigenetic diversity of Kaposi's sarcoma-associated herpesvirus.

Spontaneous lytic reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) occurs at a low rate in latently infected cells in disease and culture. This suggests imperfect epigenetic maintenance of viral transcription programs, perhaps due to variability in chromatin structure at specific loci across the population of KSHV episomal genomes. To characterize this locus-specific chromatin structural diversity, we used MAPit single-molecule footprinting, which simultaneously maps endogenous CG methylation and accessibility to M.CviPI at GC sites. Diverse chromatin structures were detected at the LANA, RTA and vIL6 promoters. At each locus, chromatin ranged from fully closed to fully open across the population. This diversity has not previously been reported in a virus. Phorbol ester and RTA transgene induction were used to identify chromatin conformations associated with reactivation of lytic transcription, which only a fraction of episomes had. Moreover, certain chromatin conformations correlated with CG methylation patterns at the RTA and vIL6 promoters. This indicated that some of the diverse chromatin conformations at these loci were epigenetically distinct. Finally, by comparing chromatin structures from a cell line infected with constitutively latent virus, we identified products of lytic replication. Our findings show that epigenetic drift can restrict viral propagation by chromatin compaction at latent and lytic promoters.

HITS-CLIP and PAR-CLIP advance viral miRNA targetome analysis.

MiRNAs regulate gene expression by binding predominantly to the 3' untranslated region (UTR) of target transcripts to prevent their translation and/or induce target degradation. In addition to the more than 1200 human miRNAs, human DNA tumor viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) encode miRNAs. Target predictions indicate that each miRNA targets hundreds of transcripts, many of which are regulated by multiple miRNAs. Thus, target identification is a big challenge for the field. Most methods used currently investigate single miRNA-target interactions and are not able to analyze complex miRNA-target networks. To overcome these challenges, cross-linking and immunoprecipitation (CLIP), a recently developed method to study direct RNA-protein interactions in living cells, has been successfully applied to miRNA target analysis. It utilizes Argonaute (Ago)-immunoprecipitation to isolate native Ago-miRNA-mRNA complexes. In four recent publications, two variants of the CLIP method (HITS-CLIP and PAR-CLIP) were utilized to determine the targetomes of human and viral miRNAs in cells infected with the gamma-herpesviruses KSHV and EBV, which are associated with a number of human cancers. Here, we briefly introduce herpesvirus-encoded miRNAs and then focus on how CLIP technology has largely impacted our understanding of viral miRNAs in viral biology and pathogenesis.

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 KSHV miRNA 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/EBPß. 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.

A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 viral microRNAs (miRNAs) that are expressed during latency. Research into KSHV miRNA function has suffered from a lack of genetic systems to study viral miRNA mutations in the context of the viral genome. We used the Escherichia coli Red recombination system together with a new bacmid background, BAC16, to create mutants for all known KSHV miRNAs. The specific miRNA deletions or mutations and the integrity of the bacmids have been strictly quality controlled using PCR, restriction digestion, and sequencing. In addition, stable viral producer cell lines based on iSLK cells have been created for wildtype KSHV, for 12 individual miRNA knock-out mutants (ΔmiR-K12-1 through -12), and for mutants deleted for 10 of 12 (ΔmiR-cluster) or all 12 miRNAs (ΔmiR-all). NGS, in combination with SureSelect technology, was employed to sequence the entire latent genome within all producer cell lines. qPCR assays were used to verify the expression of the remaining viral miRNAs in a subset of mutants. Induction of the lytic cycle leads to efficient production of progeny viruses that have been used to infect endothelial cells. Wt BAC16 and miR mutant iSLK producer cell lines are now available to the research community.

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 miR­K12-11 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.

Leveraging biological replicates to improve analysis in ChIP-seq experiments.

ChIP-seq experiments identify genome-wide profiles of DNA-binding molecules including transcription factors, enzymes and epigenetic marks. Biological replicates are critical for reliable site discovery and are required for the deposition of data in the ENCODE and modENCODE projects. While early reports suggested two replicates were sufficient, the widespread application of the technique has led to emerging consensus that the technique is noisy and that increasing replication may be worthwhile. Additional biological replicates also allow for quantitative assessment of differences between conditions. To date it has remained controversial about how to confirm peak identification and to determine signal strength across biological replicates, particularly when the number of replicates is greater than two. Using objective metrics, we evaluate the consistency of biological replicates in ChIP-seq experiments with more than two replicates. We compare several approaches for binding site determination, including two popular but disparate peak callers, CisGenome and MACS2. Here we propose read coverage as a quantitative measurement of signal strength for estimating sample concordance. Determining binding based on genomic features, such as promoters, is also examined. We find that increasing the number of biological replicates increases the reliability of peak identification. Critically, binding sites with strong biological evidence may be missed if researchers rely on only two biological replicates. When more than two replicates are performed, a simple majority rule (>50% of samples identify a peak) identifies peaks more reliably in all biological replicates than the absolute concordance of peak identification between any two replicates, further demonstrating the utility of increasing replicate numbers in ChIP-seq experiments.

γ-Herpesvirus-encoded miRNAs and their roles in viral biology and pathogenesis.

To date, more than 200 viral miRNAs have been identified mostly from herpesviruses and this rapidly evolving field has recently been summarized in a number of excellent reviews (see [1,2]). Unique to γ-herpesviruses, like Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus, is their ability to cause cancer. Here, we discuss γ-herpesvirus-encoded miRNAs and focus on recent findings which support the hypothesis that viral miRNAs directly contribute to pathogenesis and tumorigenesis. The observations that KSHV mimics a human tumorigenic miRNA (hsa-miR-155), which is induced in EBV-infected cells and required for the survival of EBV-immortalized cells, lead to a number of studies demonstrating that perturbing this pathway induces B cell proliferation in vivo and immortalization of human B cells in vitro. Secondly, the application of state of the art ribonomics methods to globally identify viral miRNA targets in virus-infected tumor cells provides a rich resource to the KSHV and EBV fields and largely expanded our understanding on how viral miRNAs contribute to viral biology.