EBV and human microRNAs co-target oncogenic and apoptotic viral and human genes during latency.

Epstein-Barr virus (EBV) controls gene expression to transform human B cells and maintain viral latency. High-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) identified mRNA targets of 44 EBV and 310 human microRNAs (miRNAs) in Jijoye (Latency III) EBV-transformed B cells. While 25% of total cellular miRNAs are viral, only three viral mRNAs, all latent transcripts, are targeted. Thus, miRNAs do not control the latent/lytic switch by targeting EBV lytic genes. Unexpectedly, 90% of the 1664 human 3'-untranslated regions targeted by the 12 most abundant EBV miRNAs are also targeted by human miRNAs via distinct binding sites. Half of these are targets of the oncogenic miR-17∼92 miRNA cluster and associated families, including mRNAs that regulate transcription, apoptosis, Wnt signalling, and the cell cycle. Reporter assays confirmed the functionality of several EBV and miR-17 family miRNA-binding sites in EBV latent membrane protein 1 (LMP1), EBV BHRF1, and host CAPRIN2 mRNAs. Our extensive list of EBV and human miRNA targets implicates miRNAs in the control of EBV latency and illuminates viral miRNA function in general.

Comprehensive analysis of Rhesus lymphocryptovirus microRNA expression.

Rhesus lymphocryptovirus (rLCV) and Epstein-Barr virus (EBV) are closely related gammaherpesviruses that infect and cause disease in rhesus monkeys and humans, respectively. Thus, rLCV is an important model system for EBV pathogenesis. Both rLCV and EBV express microRNAs (miRNAs), several conserved in sequence and genomic location. We have applied deep sequencing technology to obtain an inventory of rLCV miRNA expression in latently rLCV-infected monkey B cells. Our data confirm the presence of all previously identified mature rLCV miRNAs and have resulted in the discovery of 21 new mature miRNAs arising from previously identified precursor miRNAs (pre-miRNAs), as well as two novel pre-miRNAs (rL1-34 and rL1-35) that together generate four new mature miRNAs. Thus, the total number of rLCV-encoded pre-miRNAs is 35 and the total number of rLCV mature miRNAs is 68, the most of any virus examined. The exact 5' and 3' ends of all mature rLCV miRNAs were pinpointed, many showing marked sequence and length heterogeneity that could modulate function. We further demonstrate that rLCV mature miRNAs associate with Argonaute proteins in rLCV-infected B cells.

Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP.

Translational profiling methodologies enable the systematic characterization of cell types in complex tissues, such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy for profiling CNS cell types in a spatiotemporally restricted fashion by engineering a Cre-dependent adeno-associated virus expressing an EGFP-tagged ribosomal protein (AAV-FLEX-EGFPL10a) to access translating mRNAs by translating ribosome affinity purification (TRAP). We demonstrate the utility of this AAV to target a variety of genetically and anatomically defined neural populations expressing Cre recombinase and illustrate the ability of this viral TRAP (vTRAP) approach to recapitulate the molecular profiles obtained by bacTRAP in corticothalamic neurons across multiple serotypes. Furthermore, spatially restricting adeno-associated virus (AAV) injections enabled the elucidation of regional differences in gene expression within this cell type. Altogether, these results establish the broad applicability of the vTRAP strategy for the molecular dissection of any CNS or peripheral cell type that can be engineered to express Cre.