Titolo della tesi: Generation of a cellular model to identify small molecules active at inhibiting the anti-interferon function of the Zaire Ebola Virus protein Vp35
Ebola virus (EBOV) is responsible for the Ebola virus disease (EVD), a severe hemorrhagic fever possessing one of the highest mortality rate (up to 90% in humans). To date, the infection is mainly prevalent in areas of Central and Western Africa. The periodic re-emergence of such epidemics depends on the presence of animal reservoirs of the virus such as fruit bats belonging to the Pteropodidae family. The epidemic that lasted from 2014 to 2016 was the largest EBOV epidemic ever, causing more cases and deaths than all previous epidemics combined; for this reason, the World Health Organization (WHO) declared a state of international emergency in 2014, as there was a real risk for the spreading of the infection worldwide. Hence the need to find effective and specific drugs and vaccines to combat or prevent this disease. This work aimed to create a cellular biotechnological platform, suitable for high throughput screening of small molecules, useful to test compounds potentially capable of inhibiting the anti-IFN-I function of the VP35 protein of the EBOV. This protein is able to inhibit the production, by the host cell, of IFN-I, thus allowing the virus to evade the innate immune system. Therefore, a first engineering of K562 cells was performed, in order to express a reporter construct that allows to indirectly measure, through the Luciferase assay, the production of IFN-beta stimulated by the constitutive expression of a dsRNA. A second engineering was then carried out, in order to generate two cell lines, a control and one expressing the virus protein VP35. On this platform, a screening of 24 small molecules, selected on the basis of data in the literature that indicated the antiviral potential of molecules belonging to the classes of antidepressant, antipsycotic, antimuscarinic, or antihistaminic drugs, albeit with different mechanisms, was carried out at two distinct concentrations (10μM and 50 μM) and at two different times points (2h and 24h). The experiments revealed the efficacy of 6 compounds at inhibiting the anti-IFN activity of the viral protein VP35 from 34% to 63%, with imipramine and amitriptyline showing the best inhibitory activities. The ability, of the 6 compounds identified, to determine an increase in the expression of the IFN-beta gene was then confirmed by Real Time RT-PCR. K562 cells expressing VP35 have a reduced nuclear localization of the transcription factor IRF3, even in its hyperphosphorylated form, crucial for the induction of IFN-I production, compared to control cells. For the characterization of the mechanism of action of the 6 identified compounds, their ability to restore the nuclear levels of IRF3 and hyperphosphorylated IRF3 in cells expressing VP35 was then analyzed by western blotting. Five of these compounds restored, at different extent, the nuclear levels of IRF3 and its hyperphosphorylated form, with imipramine and amitriptyline showing the best increase in both, IFN-beta mRNA and IRF-3 nuclear localization.