Identification and characterization of viral MicroRNAs

MicroRNAs (miRNAs) are small RNAs that play important roles in the regulation of gene expression. First described as posttranscriptional gene regulators in eukaryotic hosts, virus-encoded miRNAs were later uncovered. Fundamental questions remain about which viruses encode miRNAs and what are the functions of these miRNAs. In this work we develop methods to identify virally encoded miRNAs and begin an initial characterization of these miRNAs. First, we identify new miRNA genes in both subfamilies of the retroviruses using a combined computational and synthetic approach. Previously, readily detectable, evolutionarily conserved natural miRNAs have only been identified in viruses with DNA genomes. Combined with the fact that most miRNAs are generated from endonucleolytic cleavage of longer transcripts, this finding has led to a common conception that naturally occurring viruses with RNA genomes, including retroviruses, will not encode miRNAs to avoid unproductive cleavage of their genomes or mRNAs. Here we demonstrate that the bovine leukemia virus (BLV), a deltaretrovirus, and simian foamy virus (SFV), a spumaretrovirus, encode conserved clusters of miRNAs that are transcribed by RNA polymerase III. Characterization of the BLV and SFV miRNAs reveal novel non- canonical miRNA biogenesis pathways. In addition, these viruses encode analogs of host miRNAs including miR-29, miR-132, and miR-155. Strikingly, some unrelated herpesviruses also tap into these same regulatory pathways, implying relevance to a broad range of viruses. Second, we add Anelloviridae to the growing list of virus families that encode miRNAs and suggest that miRNA-mediated immune evasion may contribute to the pervasiveness associated with some of the torque teno viruses. Finally, we sought to determine if the recently identified WU polyomavirus (WUV) encodes miRNAs. Using high-throughput sequencing we identified novel small RNAs encoded by WUV. Northern blot analysis was consistent with a larger precursor RNA being the predominant species, and analysis of the homologous region of the related KI polyomavirus revealed a similar ncRNA gene. Bioinformatic searches of existing sequence databases led to the identification of similar genes in human bocaviruses. All of these newly identified viral ncRNAs share key characteristics with the adenovirus VA RNAs.