Interaction studies between cellular proteins and viral proteins of the new coronavirus 2019 (SARS-CoV-2)

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), as well as SARS-CoV from 2003 and MERS-CoV from 2012, is part of the genus Betacoronavirus of the order Nidovirales and is currently the virus causing the pandemic called COVID- 19 (Coronavirus disease 2019). SARS-CoV-2 causes a severe acute respiratory syndrome or SARS, characterized by fever, cough, fatigue and severe cases of pneumonia, having affected more than 1.8 million people worldwide so far. Three of the virus's structural proteins, proteins E, M and N, are responsible for assembling the viral particle, among other processes. Protein N has a high affinity for viral RNA, forming the viral nucleocapsid. The membrane proteins E and M are responsible for the morphology of the virion and for interactions both with the protein S, which recognizes the cellular receptor, and with the viral nucleocapsid. The replication process of coronaviruses is highly regulated, involving different actions of the non-structural and structural proteins of the virus. However, little is known about which cellular proteins are involved in the assembly of the viral particles of the coronaviruses. This project aims to identify the interactome of SARS-CoV-2 E, M and N proteins in human cells, understanding their molecular interactions with cellular proteins and the cellular pathways that this virus benefits to complete its replication cycle. For that, we will do the optimization of the genes that encode E, M and N for expression in human cells, followed by immunoprecipitation and characterization by mass spectrometry of interacting cellular proteins. Once validated, such interactions will be challenged with pharmacological interventions in vitro, in order to verify their potential to block or interfere with viral replication. These compounds will finally be tested in an in vitro culture model of human cells infected with SARS-CoV-2, analyzing viral load, cytopathic effect and molecular changes in the cells. The present research project aims, therefore, to contribute to a better understanding of the molecular and cellular mechanisms associated with the replication of SARS-CoV-2 in human cells and to the rationalization of potential future therapies against COVID-19. (AU)