Showing papers by "Bryan R. Cullen published in 2013"

MicroRNAs as mediators of viral evasion of the immune system.

Abstract: Cellular microRNAs serve key roles in the post-transcriptional regulation of almost every cellular gene-regulatory pathway, and it therefore is not surprising that viruses have found ways to subvert this process. Several viruses encode microRNAs that directly downregulate the expression of factors of the innate immune system, including proteins involved in promoting apoptosis and recruiting effector cells of the immune system. Viruses have also evolved the ability to downregulate or upregulate the expression of specific cellular miRNAs to enhance their replication. This Review provides an overview of the present knowledge of the complex interactions of viruses with the microRNA machinery of cells.

In-Depth Analysis of the Interaction of HIV-1 with Cellular microRNA Biogenesis and Effector Mechanisms

Abstract: The question of how HIV-1 interfaces with cellular microRNA (miRNA) biogenesis and effector mechanisms has been highly controversial. Here, we first used deep sequencing of small RNAs present in two different infected cell lines (TZM-bl and C8166) and two types of primary human cells (CD4 + peripheral blood mononuclear cells [PBMCs] and macrophages) to unequivocally demonstrate that HIV-1 does not encode any viral miRNAs. Perhaps surprisingly, we also observed that infection of T cells by HIV-1 has only a modest effect on the expression of cellular miRNAs at early times after infection. Comprehensive analysis of miRNA binding to the HIV-1 genome using the photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) technique revealed several binding sites for cellular miRNAs, a subset of which were shown to be capable of mediating miRNA-mediated repression of gene expression. However, the main finding from this analysis is that HIV-1 transcripts are largely refractory to miRNA binding, most probably due to extensive viral RNA secondary structure. Together, these data demonstrate that HIV-1 neither encodes viral miRNAs nor strongly influences cellular miRNA expression, at least early after infection, and imply that HIV-1 transcripts have evolved to avoid inhibition by preexisting cellular miRNAs by adopting extensive RNA secondary structures that occlude most potential miRNA binding sites. IMPORTANCE MicroRNAs (miRNAs) are a ubiquitous class of small regulatory RNAs that serve as posttranscriptional regulators of gene expression. Previous work has suggested that HIV-1 might subvert the function of the cellular miRNA machinery by expressing viral miRNAs or by dramatically altering the level of cellular miRNA expression. Using very sensitive approaches, we now demonstrate that neither of these ideas is in fact correct. Moreover, HIV-1 transcripts appear to largely avoid regulation by cellular miRNAs by adopting an extensive RNA secondary structure that occludes the ability of cellular miRNAs to interact with viral mRNAs. Together, these data suggest that HIV-1, rather than seeking to control miRNA function in infected cells, has instead evolved a mechanism to become largely invisible to cellular miRNA effector mechanisms.

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

Abstract: 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. We experimentally identified miR-17∼92 target genes by PAR-CLIP and validated select target genes in miR-17∼92 transgenic mice. These analyses demonstrate that miR-17∼92 drives lymphomagenesis by suppressing the expression of multiple negative regulators of the PI3K and NFκB pathways and by inhibiting the mitochondrial apoptosis pathway. Accordingly, miR-17∼92-driven lymphoma cells exhibited constitutive activation of the PI3K and NFκB pathways and chemical inhibition of either pathway reduced tumour size and prolonged the survival of lymphoma-bearing mice. These findings establish miR-17∼92 as a powerful cancer driver that coordinates the activation of multiple oncogenic pathways, and demonstrate for the first time that chemical inhibition of miRNA downstream pathways has therapeutic value in treating cancers caused by miRNA dysregulation.

Mutational Inactivation of Herpes Simplex Virus 1 MicroRNAs Identifies Viral mRNA Targets and Reveals Phenotypic Effects in Culture

Abstract: Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, expresses several viral microRNAs (miRNAs). These, along with the latency-associated transcript, represent the only viral RNAs detectable in latently infected neuronal cells. Here, for the first time, we analyze which HSV-1 miRNAs are loaded into the RNA-induced silencing complex (RISC), the key effector of miRNA function. Only 9 of the 17 reported HSV-1 miRNAs, i.e., miR-H1 to miR-H8 plus miR-H11, were found to actually load into the RISC. Surprisingly, this analysis also revealed that HSV-1 miRNAs loaded into the RISC with efficiencies that differed widely; <1% of the miR-H1-3p miRNA detectable in HSV-1-infected cells was loaded into the RISC. Analysis of HSV-1 mutants individually lacking the viral miR-H2, miR-H3, or miR-H4 miRNA revealed that loss of these miRNAs affected the rate of replication of HSV-1 in neuronal cells but not in fibroblasts. Analysis of mRNA and protein expression, as well as assays mapping viral miRNA binding sites in infected cells, showed that endogenous HSV-1 miR-H2 binds to viral ICP0 mRNA and inhibits its expression, while endogenous miR-H4 inhibits the expression of the viral ICP34.5 gene. In contrast, no viral mRNA target for miR-H3 could be detected, even though miR-H3, like miR-H4, is perfectly complementary to ICP34.5 mRNA. Together, these data demonstrate that endogenous HSV-1 miRNA expression can significantly alter viral replication in culture, and they also identify two viral mRNA targets for miR-H2 and miR-H4 that can partially explain this phenotype.

MicroRNA target site identification by integrating sequence and binding information

Abstract: 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. It considerably outperforms sequence-only approaches and quantifies the prevalence of noncanonical binding modes.

How do viruses avoid inhibition by endogenous cellular microRNAs

Abstract: MicroRNAs (miRNAs) are an extensive family of small regulatory RNAs that function by binding to complementary mRNAs, primarily in the 3′ untranslated region (3′UTRs), and then inhibiting their expression by reducing mRNA translation and/or stability [1]. MiRNAs are initially transcribed as long pri-miRNAs, which are sequentially processed by the RNase III enzymes Drosha, in the nucleus, and Dicer, in the cytoplasm, to generate the mature, ∼22-nt miRNA [2]. This is then loaded into the RNA Induced Silencing Complex (RISC), which consists minimally of one of the four mammalian Argonaut proteins, Ago1 to Ago4, as well as a member of the GW182 protein family. MiRNAs function as guide RNAs to target RISC to complementary mRNA sequences on specific mRNA 3′UTRs. Analysis has revealed that complementarity to nucleotides 2 through 8 of the miRNA, the so-called seed region, is particularly important for effective RISC recruitment [1], although non-canonical sites, with incomplete seed complementarity, have also been reported [3]. Importantly, RISC recruitment to target sites that are occluded by RNA secondary structure or bound proteins is very inefficient [4].

A Cluster of Virus-Encoded MicroRNAs Accelerates Acute Systemic Epstein-Barr Virus Infection but Does Not Significantly Enhance Virus-Induced Oncogenesis In Vivo

Abstract: Over 90% of the adult human population is chronically infected with the Epstein-Barr virus (EBV), an oncogenic herpesvirus. EBV primarily infects naive human B cells and persists latently in memory B cells. Most individuals experience an asymptomatic infection that is effectively controlled by the adaptive immune response. However, EBV-associated lymphomas can develop in immunocompromised individuals. These tumors typically express all nine EBV latent proteins (latency III). Latency III is also associated with the expression of three precursor microRNAs (miRNAs) located within the EBV BHRF1 gene locus. The role of these BHRF1 miRNAs was unclear until recent in vitro studies demonstrated that they cooperate to enhance virus-induced B cell transformation and decrease the antigenic load of virus-infected cells, indicating that the BHRF1 miRNA cluster may serve as a novel therapeutic target for the treatment of latency III EBV-associated malignancies. However, to date, it is not known if BHRF1 miRNAs enhance virus-induced oncogenesis and/or immune evasion of EBV in vivo. To understand the in vivo contribution of the BHRF1 miRNA cluster to EBV infection and EBV-associated tumorigenesis, we monitored EBV infection and assessed tumor formation in humanized mice exposed to wild-type virus and a viral mutant (Δ123) that lacks all three BHRF1 miRNAs. Our results demonstrate that while the BHRF1 miRNAs facilitate the development of acute systemic EBV infection, they do not enhance the overall oncogenic potential of EBV in vivo.