Identification of virus-encoded microRNAs in divergent Papillomaviruses

MicroRNAs (miRNAs) are small RNAs that regulate diverse biological processes including multiple aspects of the host-pathogen interface. Consequently, miRNAs are commonly encoded by viruses that undergo long-term persistent infection. Papillomaviruses (PVs) are capable of undergoing persistent infection, but as yet, no widely-accepted PV-encoded miRNAs have been described. The incomplete understanding of PV-encoded miRNAs is due in part to lack of tractable laboratory models for most PV types. To overcome this, we have developed miRNA Discovery by forced Genome Expression (miDGE), a new wet bench approach to miRNA identification that screens numerous pathogen genomes in parallel. Using miDGE, we screened over 73 different PV genomes for the ability to code for miRNAs. Our results show that most PVs are unlikely to code for miRNAs and we conclusively demonstrate a lack of PV miRNA expression in cancers associated with infections of several high risk HPVs. However, we identified five different high-confidence or highly probable miRNAs encoded by four different PVs (Human PVs 17, 37, 41 and a Fringilla coelebs PV (FcPV1)). Extensive in vitro assays confirm the validity of these miRNAs in cell culture and two FcPV1 miRNAs are further confirmed to be expressed in vivo in a natural host. We show that miRNAs from two PVs (HPV41 & FcPV1) are able to regulate viral transcripts corresponding to the early region of the PV genome. Combined, these findings identify the first canonical PV miRNAs and support that miRNAs of either host or viral origin are important regulators of the PV life cycle

Engineered Versions of Granzyme B and Angiogenin Overcome Intrinsic Resistance to Apoptosis Mediated by Human Cytolytic Fusion Proteins

The use of therapies based on antibody fusion proteins for the selective elimination of tumor cells has increased markedly over the last two decades because the severe side effects associated with conventional chemotherapy and radiotherapy are reduced or even eliminated. However, the initial development of immunotoxins suffered from a number of drawbacks such as nonspecific cytotoxicity and the induction of immune responses because the components were non-human in origin. The most recent iteration of this approach is a new class of targeted human cytolytic fusion proteins (hCFPs) comprising a tumor-specific targeting component such as a human antibody fragment fused to a human effector domain with pro-apoptotic activity. Certain tumors resist the activity of hCFPs by upregulating the intracellular expression of native inhibitors, which rapidly bind and inactivate the human effector domains. Higher doses of the hCFPs are, therefore, required to improve therapeutic efficacy. To circumvent these inhibitory processes, novel isoforms of the enzymes granzyme B and angiogenin have been designed to increase their intrinsic activity and reduce their interactions with native inhibitors resulting in more potent hCFPs that can be applied at lower doses. This chapter summarizes the basic scientific knowledge that can facilitate the rational development of human enzymes with novel and beneficial characteristics, including the ability to avoid neutralization by native inhibitors