Sequence Analysis of microRNAs Encoded by Simian Lymphocryptoviruses.

Lymphocryptoviruses (LCVs) are ubiquitous gamma-herpesviruses that establish life-long infections in both humans and non-human primates (NHPs). In immunocompromised hosts, LCV infections are commonly associated with B cell disorders and malignancies such as lymphoma. In this study, we evaluated simian LCV-encoded small microRNAs (miRNAs) present in lymphoblastoid cell lines (LCLs) derived from a Mauritian cynomolgus macaque () with cyLCV-associated post-transplant lymphoproliferative disease (PTLD) as well as the viral miRNAs expressed in a baboon () LCL that harbors CeHV12.

MicroRNA-focused CRISPR/Cas9 screen identifies miR-142 as a key regulator of Epstein-Barr virus reactivation.

Reactivation from latency plays a significant role in maintaining persistent lifelong Epstein-Barr virus (EBV) infection. Mechanisms governing successful activation and progression of the EBV lytic phase are not fully understood. EBV expresses multiple viral microRNAs (miRNAs) and manipulates several cellular miRNAs to support viral infection.

MicroRNA-mediated control of Epstein-Barr virus infection and potential diagnostic and therapeutic implications.

Herpesviruses, such as Epstein-Barr virus (EBV), encode multiple viral microRNAs that are expressed throughout various infection stages. While much progress has been made in evaluating both the viral and host microRNAs (miRNAs) that are detected during infection as well as elucidating their molecular targets in vitro, our understanding of their contributions to pathogenesis in vivo, viral oncogenesis, and clinical implications for these small molecules remains limited. miRNAs are widely recognized as key regulators of global cellular processes, including apoptosis, cell differentiation, and development of immune responses.

Effects of Early Antiretroviral Therapy on the Composition and Diversity of the Fecal Microbiome of SIV-infected Rhesus Macaques ().

HIV-infected people develop reproducible disruptions in their gastrointestinal microbiota. Despite the suppression of HIV viremia via long-term antiretroviral therapy (ART), alterations still occur in gut microbial diversity and the commensal microbiota. Mounting evidence suggests these microbial changes lead to the development of gut dysbiosis-persistent inflammation that damages the gut mucosa-and correlate with various immune defects.

Rapamycin limits CD4+ T cell proliferation in simian immunodeficiency virus-infected rhesus macaques on antiretroviral therapy.

Proliferation of latently infected CD4+ T cells with replication-competent proviruses is an important mechanism contributing to HIV persistence during antiretroviral therapy (ART). One approach to targeting this latent cell expansion is to inhibit mTOR, a regulatory kinase involved with cell growth, metabolism, and proliferation. Here, we determined the effects of chronic mTOR inhibition with rapamycin with or without T cell activation in SIV-infected rhesus macaques (RMs) on ART.

Epstein-Barr Virus miR-BHRF1-3 Targets the BZLF1 3'UTR and Regulates the Lytic Cycle.

Suppression of lytic viral gene expression is a key aspect of the Epstein-Barr virus (EBV) life cycle to facilitate the establishment of latent infection. Molecular mechanisms regulating transitions between EBV lytic replication and latency are not fully understood. Here, we investigated the impact of viral microRNAs on the EBV lytic cycle.

B Cell Receptor-Responsive miR-141 Enhances Epstein-Barr Virus Lytic Cycle via FOXO3 Inhibition.

Antigen recognition by the B cell receptor (BCR) is a physiological trigger for reactivation of Epstein-Barr virus (EBV) and can be recapitulated by cross-linking of surface immunoglobulins. Previously, we identified a subset of EBV microRNAs (miRNAs) that attenuate BCR signal transduction and subsequently dampen lytic reactivation in B cells. The roles of host miRNAs in the EBV lytic cycle are not completely understood.

Multiple Viral microRNAs Regulate Interferon Release and Signaling Early during Infection with Epstein-Barr Virus.

Epstein-Barr virus (EBV), a human herpesvirus, encodes 44 microRNAs (miRNAs), which regulate many genes with various functions in EBV-infected cells. Multiple target genes of the EBV miRNAs have been identified, some of which play important roles in adaptive antiviral immune responses. Using EBV mutant derivatives, we identified additional roles of viral miRNAs in governing versatile type I interferon (IFN) responses upon infection of human primary mature B cells.

Regulation of Latency and Reactivation by Human Cytomegalovirus miRNAs.

Human cytomegalovirus (HCMV) encodes 22 mature microRNAs (miRNAs), which regulate a myriad of cellular processes, including vesicular trafficking, cell cycle progression, apoptosis, and immune evasion, as well as viral gene expression. Recent evidence points to a critical role for HCMV miRNAs in mediating latency in CD34 hematopoietic progenitor cells through modulation of cellular signaling pathways, including attenuation of TGFβ and EGFR signaling. Moreover, HCMV miRNAs can act in concert with, or in opposition to, viral proteins in regulating host cell functions.

Techniques for Characterizing Cytomegalovirus-Encoded miRNAs.

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level by binding to sites within the 3' untranslated regions of messenger RNA (mRNA) transcripts. The discovery of this completely new mechanism of gene regulation necessitated the development of a variety of techniques to further characterize miRNAs, their expression, and function. In this chapter, we will discuss techniques currently used in the miRNA field to detect, express and inhibit miRNAs, as well as methods used to identify and validate their targets, specifically with respect to the miRNAs encoded by human cytomegalovirus.

Role of IL-15 Signaling in the Pathogenesis of Simian Immunodeficiency Virus Infection in Rhesus Macaques.

Although IL-15 has been implicated in the pathogenic hyperimmune activation that drives progressive HIV and SIV infection, as well as in the generation of HIV/SIV target cells, it also supports NK and T cell homeostasis and effector activity, potentially benefiting the host. To understand the role of IL-15 in SIV infection and pathogenesis, we treated two cohorts of SIVmac239-infected rhesus macaques (RM; ), one with chronic infection, the other with primary infection, with a rhesusized, IL-15-neutralizing mAb (versus an IgG isotype control) for up to 10 wk ( = 7-9 RM per group). In both cohorts, anti-IL-15 was highly efficient at blocking IL-15 signaling in vivo, causing 1) profound depletion of NK cells in blood and tissues throughout the treatment period; 2) substantial, albeit transient, depletion of CD8 effector memory T cells (T) (but not the naive and central memory subsets); and 3) CD4 and CD8 T hyperproliferation.

Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation.

MicroRNAs (miRNAs) are post-transcriptional regulatory RNAs that can modulate cell signaling and play key roles in cell state transitions. Epstein-Barr virus (EBV) expresses >40 viral miRNAs that manipulate both viral and cellular gene expression patterns and contribute to reprogramming of the host environment during infection. Here, we identified a subset of EBV miRNAs that desensitize cells to B cell receptor (BCR) stimuli, and attenuate the downstream activation of NF-kappaB or AP1-dependent transcription.

An Epstein-Barr Virus MicroRNA Blocks Interleukin-1 (IL-1) Signaling by Targeting IL-1 Receptor 1.

Epstein-Barr virus (EBV) encodes >44 viral microRNAs (miRNAs) that are differentially expressed throughout infection, can be detected in Epstein-Barr virus (EBV)-positive tumors, and manipulate several biological processes, including cell proliferation, apoptosis, and immune responses. Here, we show that EBV BHRF1-2 miRNAs block NF-κB activation following treatment with proinflammatory cytokines, specifically interleukin-1β (IL-1β). Analysis of EBV PAR-CLIP miRNA targetome data sets combined with pathway analysis revealed multiple BHRF1-2 miRNA targets involved in interleukin signaling pathways.

Roles of Non-coding RNAs During Herpesvirus Infection.

Non-coding RNAs (ncRNAs) play essential roles in multiple aspects of the life cycles of herpesviruses and contribute to lifelong persistence of herpesviruses within their respective hosts. In this chapter, we discuss the types of ncRNAs produced by the different herpesvirus families during infection, some of the cellular ncRNAs manipulated by these viruses, and the overall contributions of ncRNAs to the viral life cycle, influence on the host environment, and pathogenesis.

Analysis of Viral and Cellular MicroRNAs in EBV-Infected Cells.

MicroRNAs are small, noncoding RNAs that posttranscriptionally regulate gene expression. The discovery of this relatively new mode of gene regulation as well as studies showing the prognostic value of viral and cellular miRNAs as biomarkers, such as in cancer progression, has stimulated the development of many methods to characterize miRNAs. EBV encodes 25 viral precursor microRNAs within its genome that are expressed during lytic and latent infection.

Japanese Macaque Rhadinovirus Encodes a Viral MicroRNA Mimic of the miR-17 Family.

Unlabelled: Japanese macaque (JM) rhadinovirus (JMRV) is a novel, gamma-2 herpesvirus that was recently isolated from JM with inflammatory demyelinating encephalomyelitis (JME). JME is a spontaneous and chronic disease with clinical characteristics and immunohistopathology comparable to those of multiple sclerosis in humans. Little is known about the molecular biology of JMRV.

EBV Noncoding RNAs.

EBV expresses a number of viral noncoding RNAs (ncRNAs) during latent infection, many of which have known regulatory functions and can post-transcriptionally regulate viral and/or cellular gene expression. With recent advances in RNA sequencing technologies, the list of identified EBV ncRNAs continues to grow. EBV-encoded RNAs (EBERs) , the BamHI-A rightward transcripts (BARTs) , a small nucleolar RNA (snoRNA) , and viral microRNAs (miRNAs) are all expressed during EBV infection in a variety of cell types and tumors.

EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that can give rise to cancers of both B-cell and epithelial cell origin. In EBV-induced cancers of epithelial origin, including nasopharyngeal carcinomas (NPCs) and gastric carcinomas, the latent EBV genome expresses very high levels of a cluster of 22 viral pre-miRNAs, called the miR-BARTs, and these have previously been shown to confer a degree of resistance to pro-apoptotic drugs. Here, we present an analysis of the ability of individual miR-BART pre-miRNAs to confer an anti-apoptotic phenotype and report that five of the 22 miR-BARTs demonstrate this ability.

Replication of many human viruses is refractory to inhibition by endogenous cellular microRNAs.

The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs.

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential.

It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression.

Evolutionary conservation of primate lymphocryptovirus microRNA targets.

Epstein-Barr virus (EBV) and rhesus lymphocryptovirus (rLCV) are closely related gammaherpesviruses in the lymphocryptovirus subgroup that express viral microRNAs (miRNAs) during latent infection. In addition to many host mRNAs, EBV miRNAs are known to target latent viral transcripts, specifically those encoding LMP1, BHRF1, and EBNA2. The mRNA targets of rLCV miRNAs have not been investigated.

MicroRNA-17~92 plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways.

MicroRNAs (miRNAs) have been broadly implicated in cancer, but their exact function and mechanism in carcinogenesis remain poorly understood. Elevated miR-17~92 expression is frequently found in human cancers, mainly due to gene amplification and Myc-mediated transcriptional upregulation. Here we show that B cell-specific miR-17~92 transgenic mice developed lymphomas with high penetrance and that, conversely, Myc-driven lymphomagenesis stringently requires two intact alleles of miR-17~92.

MicroRNA target site identification by integrating sequence and binding information.

High-throughput sequencing has opened numerous possibilities for the identification of regulatory RNA-binding events. Cross-linking and immunoprecipitation of Argonaute proteins can pinpoint a microRNA (miRNA) target site within tens of bases but leaves the identity of the miRNA unresolved. A flexible computational framework, microMUMMIE, integrates sequence with cross-linking features and reliably identifies the miRNA family involved in each binding event.