Characterization of the biogenesis and function of miRNAs encoded by diverse tumor viruses

Some eukaryotic viruses express small RNAs called microRNAs (miRNAs) to regulate host and viral gene expression. Due to their small genomic footprint, ability to regulate numerous genes, and lack of immunogenicity, miRNAs are an apt gene regulatory mechanism for viruses. Nevertheless, few viral miRNAs have been studied in vivo and the biological functions of most remain unknown. All known viral miRNAs are generated via host machinery. Most viral miRNAs mature through the canonical miRNA biogenesis pathway, whereas a few viruses generate miRNAs using noncanonical miRNA mechanisms. The noncanonical biogenesis mechanisms and the variety of miRNA alleles associated with some viruses provide powerful tools for probing mammalian small RNA biology. In this dissertation, I analyze the biogenesis and function of miRNAs encoded by diverse tumor viruses. In chapter 2, I utilize the Simian virus 40 (SV40) primary miRNA (pri-miRNA) as a system to provide new mechanistic insights relevant to canonical pri-miRNA processing. This work reveals that multiple pri-miRNA structures coordinate processing by the Microprocessor complex. In chapter 3, I characterize the biogenesis of noncanonical miRNAs encoded by bovine leukemia virus (BLV), a deltaretrovirus. This work demonstrates that the BLV pre-miRNAs are directly transcribed by RNA polymerase III from proviral genomes, circumventing the requirement of mRNA cleavage for miRNA production. In chapter 4, using BLV and Adenoviral miRNAs, I demonstrate that dual-specificity phosphatase 11 (DUSP11) promotes accumulation and activity of small RNAs derived from diverse 5´-triphosphorylated precursors in the RNA-induced silencing complex (RISC). In chapter 5, I develop a novel method to express short hairpin RNAs (shRNAs) using the architecture of the BLV miRNA genes, thereby decreasing the template space required for shRNA expression. This work applies to gene silencing strategies where smaller gene cassettes are desirable. In chapter 6, I report that the miRNAs encoded by murine polyomavirus (MuPyV) are not required for viral persistence. Instead, the MuPyV miRNAs promote viral shedding during the acute phase of infection in vivo. This work provides a fundamental understanding of the functional role of polyomavirus-encoded miRNAs. Combined, this work advances the fields of experimental gene silencing, small RNA biology, and virus-host interactions.