The evolutionary emergence of microRNA (miRNA) biology coincided with multicellularity and arose independently in plants and animals. Similar to the antiviral defenses of plants, worms, and arthropods, miRNAs are utilized by most eukaryotic species as a means of imposing post-transcriptional silencing. This regulatory mechanism is distinct from the antiviral RNAi pathways observed in plants, worms, and arthropods. Unlike the biology used in antiviral defenses, miRNAs are genome-encoded and interact with mRNA with imperfect complementarity. This mismatch prevents small RNA-mediated cleavage by the RNA-induced silencing complex (RISC), and as a result, miRNAs are considered fine-tuners of host biology. However, should a perfect binding site for a given miRNA be introduced into a desired transcript, full cleavage and potent silencing can be restored.

We first adapted this idea as a vaccine platform that would allow a virus to be attenuated in one species but replicate to high titers in another based on the exploitation of host-specific miRNA profiles. Building on this idea, we later adapted it as a means of molecular biocontainment to generate viruses that could only replicate in a single model system. The potency of this targeting can be visualized by comparing a Sendai virus encoding GFP that includes a scrambled 3’UTR (denoted SeV-GFP) to one targeted by host miRNAs (SeV-GFP-T). Comparing the expression of the viral hemagglutinin-neuraminidase (HN) protein to GFP illustrates how potent miRNA targeting can be in the presence of a functional miRNA system. Despite a complete loss of GFP in wild type cells, fluorescence is visible when SeV-GFP-T infects cells lacking either Dicer (Dcr-/-) or Ago2 (Ago2-/-)—the only Argonaute protein capable of cleaving its target when perfect complementarity is achieved between a miRNA and its target.

We also use miRNA-based targeting to compare the genetic plasticity of viruses and determine how distinct viral families adapt to the same selective pressure. These efforts not only demonstrated that positive-sense single-stranded RNA (+ssRNA) viruses undergo rapid homologous recombination to divert targeting, but they also revealed a unique aspect of our own biological defense systems. In contrast to +ssRNA viruses which rapidly excise the targeting cassette, miRNA-based targeting of negative-sense single-stranded RNA (-ssRNA) viruses completely eliminated any expression of the virus for up to six days post-infection. However, beyond this point, we observe consistent escape of the virus as a result of multiple A-to-G substitutions across all of the miRNA targets. This viral escape is lost in cells deficient in the double-stranded RNA deaminase (ADAR1) and in those lacking Ago1/3/4, unless they are reconstituted. These results suggest that ADAR1 may utilize miRNA biology to perform its recently described function of eliminating double-stranded RNA production from the cell.