Project I: Development of a virus-free cell assay for the evaluation of viral protease inhibitors Project II: Role of microRNA hsa-mir-1307-3p in genome instability

One of the most promising strategies to counteract viral infections is the identification of molecular targets that play essential roles in viral life cycle. The development of new inhibitors involves in vitro screening of libraries of compounds, evaluating their activity against recombinant enzymes through biochemical assays. Hit compounds are then tested in cell-based systems to assess their cytotoxicity and antiviral efficacy. However, this step requires handling live viruses, which involves high biosafety levels facilities that are beyond the reach of most laboratories. In this PhD thesis, we have developed and optimized a novel virus-free, luminometric cell-based assay for evaluating inhibitors targeting two clinically relevant proteases: Mpro, the main protease of SARS-CoV-2, and NS3, the serine protease of West Nile Virus. In our assay, we engineered the NanoBiT luciferase joining the two subunits of the enzyme with a flexible spacer containing a cleavage site specific either for Mpro or NS3. In absence of the protease, the intact luciferase emits a bioluminescent signal, while expression of the viral protease leads to cleavage of the spacer, resulting in a loss of luciferase activity. In this system, the presence of a protease inhibitor prevents the cleavage of the target luciferase, thereby restoring the luminescent signal proportionally to the inhibitor’s potency. Our assay was used to evaluate two well-characterized Mpro inhibitors, GC376 and Nirmatrelvir and a small library of novel compounds. Our virus-free systems provide a powerful and versatile platform for the screening and characterization of protease inhibitors, with potential applications in both academic research and pharmaceutical development. MicroRNAs (miRNAs) are key regulators of gene expression, and their dysregulation contributes to both tumorigenesis and cellular response to viral infections. Due to their involvement in cancer progression and viral infection, miRNAs are promising therapeutic targets. Among them, the human miRNA hsa-miR-1307-3p has been reported to act as an oncogenic miRNA. The aim of this thesis was to investigate the role of hsa-miR-13073p in genomic stability and its involvement in regulation of cellular stress response. We demonstrated that inhibition of hsa-miR-1307-3p affects cell viability, activates DNA damage response markers, and decreases mitotic activity. To further investigate the role of hsa-miR-1307-3p, two stable cell lines were generated: one overexpressing hsa-miR1307-3p and another carrying a non-targeting control sequence. Cells treated with the hsa-miR-1307-3p inhibitor in combination with etoposide, a potent topoisomerase II inhibitor that induces double-strand breaks, showed a synergistic cytotoxic effect, supporting the therapeutic potential of targeting hsa-miR-1307-3p in combination with DNA-damaging agents. In silico analysis predicted high‑confidence binding sites for hsa-miR-1307-3p within the 5’‑UTR of three proteins involved in DNA:RNA hybrids metabolism: DDX5, DDX1, and RNaseH2B. We demonstrated that overexpression of hsa-miR-1307-3p significantly reduced the expression of DDX5 and RNaseH2B, suggesting a role in DNA:RNA hybrids processing. Immunofluorescence assays confirmed this effect, showing an accumulation of DNA:RNA hybrids in cells overexpressing hsa-miR-1307-3p, while inhibition of the miRNA decreased hybrids levels. Finally, flow cytometry analysis excluded cell cycle arrest as the underlying cause of altered protein expression, confirming a direct regulatory role for hsa-miR-1307-3p. Overall, this work identifies hsa-miR-1307-3p as a possible regulator of genomic stability and DNA damage response. These findings expand our understanding of its oncogenic role and support its potential as a therapeutic target in combination with genotoxic agents.