Characterization of SARS-CoV-2 protease NSP3 and NSP5 degradome.

Infectious diseases are serious threats to human health and economy in both developed and non-developed countries. They are the cause of approximately half of the death toll worldwide. Recently, a severe acute respiratory syndrome, named COVID-19 (coronavirus disease 2019), has spread across the globe and has been found to be caused by a novel strain of coronaviruses (SARS-CoV-2). As increased levels of plasma chemokines and, cytokines and an uncontrolled influx of inflammatory cells were observed in lethal cases, it was concluded that the severity of the infection corresponded with the imbalanced host immunity against the virus. The key question of the project is how SARS-CoV-2 open reading frames (ORFs) modulate the innate immune responses of the host cells. In particular, we focus on two viral non-structural proteins (NSPs) that are NSP3 and NSP5, two virally encoded proteases which are responsible of the polyprotein precursors (pp1a and pp1ab) processing from which all non-structural proteins are originated. The spotlights are on the potential role that the proteases can have in SARS-CoV-2 viral spread and infection. We plan to use a degradomics approach to characterize the entire set of their substrates to understand how SARS-CoV-2 NSP3 and NSP5 modulate innate immunity of monocytic TPH-1 cells, and pneumocytic A549 cells. In particular, the Lenti-X Tet-On 3G Inducible Expression (Takara Bio, USA) was used to clone the viral proteases under the control of a TRE3G promoter in the responder plasmid. Subsequently, cells have been transfected with TET-ON inducible plasmid. After the selection, the cells that express the Tet-On 3G transactivator protein and GOI-encoding responder plasmid will express the GOI only in the presence of doxycycline (Dox). We chose an inducible expression system of these proteases in the cells in order to better control their function/activity and avoid unspecific cuts and cell death induction. In order to test the inducibility, a luciferase assay was performed to evaluate the bioluminescent signal produced after the electroporation with pLVX-TRE3G-Luc Vector (responsive vector) of the cells previously transfected with pLVX-Tet3G (regulator) inducible plasmid. Once the Lenti-X Tet-On 3G Inducible Expression System will be ready to express the proteases in the cells in a Doxycycline-dependent manner, the heart of the project will be reached with the degradomics analysis and degradome characterization (repertoires of proteases and protease-substrates), in the view that NSP3 and NSP5 viral proteases are powerful drug target. In fact, new discoveries can support the development of therapeutical approaches, for example taking advantage of the structurally conserved substrate-binding region to develop protease inhibitors.