Studying Coronavirus–Host Protein Interactions

Abstract

To understand the molecular mechanisms of viral replication and pathogenesis, it is necessary to establish the virus-host protein interaction networks. The yeast two-hybrid system is a powerful proteomic approach to study protein-protein interactions. After the identification of specific cellular factors interacting with the target viral protein using the yeast two-hybrid screening system, co-immunoprecipitation and confocal microscopy analyses are often used to verify the virus-host protein interactions in cells. Identification of the cellular factors required for viral survival or eliminating virus infected cells could help scientists develop more effective antiviral drugs. Here we summarize a standard protocol used in our lab to study the coronavirus-host protein interactions, including yeast two-hybrid screening, co-immunoprecipitation, and immunofluorescence microscopy analyses.

Cellular PSMB4 Protein Suppresses Influenza A Virus Replication through Targeting NS1 Protein

“…The yeast two-hybrid screening system was purchased from Clontech Laboratories (Mountain View, CA, USA). The screening procedures were performed following the manufacturer’s instructions and our previous protocols [ 15 ]. The IAV NS1 gene was cloned into the pBD-GAL4 Cam phagemid vector (Agilent Technology, Palo Alto, CA, USA).…”

“…Plasmid DNA extracted from the individual yeast clones grown in -W-L-H + G + 3AT selection plates was transformed into E. coli (DH5α) competent cells separately. Then, cDNA inserts in pACT2 were extracted from these bacteria grown in ampicillin selection plates and identified by sequencing [ 15 ].…”