Characterization of host substrate of SARS-CoV-2 NSP3 and NSP5 using a degradome approach

INTRODUCTION: The COVID-19 disease emerged in December 2019 and caused a global threat to the global population. The causative virus, SARS-CoV-2 belongs to the Coronaviridae virus family. The SARS-CoV-2, once attached, internalizes its virus particle into the cell. Then, the virus particle opens and releases its RNA genome. This viral RNA is directly translated into a single long polypeptide (pp1a and pp1ab) that must be proteolytically processed to produce the set of viral proteins among which non-structural protein NSP3 (papain like proteases) and NSP5 (main protease 3-chymotrypsin-like) are the only two viral proteases. These proteases are essential to the viral replication and life cycle. The immune system antiviral response depends on the recognition of viral components and the severity of the disease is directly related to both host and virus genetic makeup. Degradome analysis allows to characterize the function of proteases by the exhaustive identification of their substrates to determine the cellular pathways upon which the proteases exert control. OBJECTIVE: we aim at better evaluating the host cells and pathogens interaction during SARS-CoV-2 infection by characterizing NSP3 and NSP5 degradomes. This degradomic analysis will better our understand SARS-CoV-2 pathophysiology and to help in developing alternative treatments more efficient against genetic variants in a long term. METHODS: we develop an inducible expression system for NSP3 and NSP5 proteases. We generated doxycycline (Dox)-inducible THP1, a monocyte cell line, and A549, a type II pneumocyte line, to investigate NSP3 and NSP5 degradome respectively in immune and pneumocyte cells, which are relevant for the pathophysiology of COVID 19. RESULTS: cells were electroporated with pLVX-Tet3G encoding for the Tet-3G transcriptional activator and selected with antibiotic. Selected cells were cloned by limiting dilution. To evaluate the Dox inducibility of the clones, a sample of each clone was transduced with pLVX TRE3G Luc, encoding for the firefly luciferase reporter under the control of the Tet-3G responsive promoter, and selected with antibiotic. Then, Firefly luciferase activity was tested with by glow luciferase assay. For each cell line, 2 Dox-inducible clones were selected, one with a medium and the other with a higher inducibility. The 4 selected clones were transfected with pLVX TREG-NSP3 and pLVX TREG-NSP5 encoding for wild type or catalytically inactive mutant forms of the proteases, selected with antibiotic and protease expression tested by Western Blot analysis. Our preliminary data suggest that we could express NSP3 and NSP5 in our inducible cell expression system. In sequence the degradome characterization will be performed.