Bearing the largest single-stranded RNA genome in nature, SARS-CoV-2 utilizes sophisticated replication/transcription complexes (RTCs), mainly composed of a network of nonstructural proteins and nucleocapsid protein, to establish efficient infection. Here, we developed an innovative interaction screening strategy based on phase separation in cellulo, namely compartmentalization of protein-protein interactions in cells (CoPIC). Utilizing CoPIC screening, we mapped the interaction network among RTC-related viral proteins. We identified a total of 47 binary interactions among 14 proteins governing replication, discontinuous transcription, and translation of coronaviruses. Further exploration via CoPIC led to the discovery of extensive ternary complexes composed of these components, which infer potential higher-order complexes. Taken together, our results present an efficient, and robust interaction screening strategy, and indicate the existence of a complex interaction network among RTC-related factors, thus opening up new opportunities to understand SARS-CoV-2 biology and develop therapeutic interventions for COVID-19.
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