key: cord-1014417-ghazal2a authors: Xu, Weifan; Pei, Gaofeng; Liu, Hongrui; Wang, Jing; Li, Pilong title: Extensive High-Order Complexes within SARS-CoV-2 Proteome Revealed by Compartmentalization-Aided Interaction Screening date: 2020-12-26 journal: bioRxiv DOI: 10.1101/2020.12.26.424422 sha: 67d8840cab579aa8fb40ff51d81b46374c0d8b2b doc_id: 1014417 cord_uid: ghazal2a 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. Establishment of robust scaffolds for phase separation in cells 105 The design strategy of CoPIC is as follows: i) a system (called scaffold) capable of 106 forming membraneless phase-separated compartments in cells is generated; ii) one abbreviated to p53 hereafter) labeled with mCherry (mCherry-p53) in cells, the mCherry 144 red fluorescence signal was enriched within the green compartments (Figures 1D, 1E) . 145 Moreover, the enrichment is MDM2-dependent as mCherry-p53 was not recruited into GFP-Nup98N-derived compartments (Figures 1F, 1G) . Similar results were obtained 147 for Hfq-GFP-FUSN-MDM2 and mCherry-p53 (Figures S1J, S1K) . 148 To further confirm that the enrichment of red fluorescence signal was due to the 149 cognate interaction between MDM2 and p53, we treated the cells co-expressing GFP-150 Nup98N-MDM2 and mCherry-p53 with Mi-773, a potent inhibitor specifically targeting 151 the MDM2/p53 interaction . Upon treatment with Mi-773, the 152 mCherry signal within the puncta immediately started to decrease and little remained by 153 46 seconds post-treatment ( Figure 1H) . Correspondingly, the ratio between the 154 intensity of mCherry over that of GFP and Pearson's correlation coefficient also 155 decreased (Figures 1I, S1L) . Notably, the rapid response to Mi-773 suggests the . We performed binary co-expression experiments to assess the interaction 175 relationship of these components using CoPIC. Consistently, the co-expression of mCherry-RBBP4 with GFP-Nup98N-SUZ12, but not that of miRFP-RBBP7 with GFP-177 Nup98N-EZH2, resulted in client enrichment in the compartments (Figures 2A, 2B, 2C 178 and 2D). 179 Further pairwise interactions between all components of PRC2 were conducted. The 180 results were consistent with the previous studies, where the interactions between EED 181 and EZH1/2, between EZH1/2 and SUZ12, and between SUZ12 and RBBP4/7 were 182 direct and all other interactions were indirect ( Figure 2E) . As a key component of 183 PRC2, SUZ12 is reported to adopt an extended conformation with the C-terminus 184 binding EZH2 and the N-terminus binding RBBP4/7 and two accessory proteins, PHF1 185 and AEBP2 (Figures 2F, 2G) . Subsequent CoPIC assays utilizing the C-terminal part of 186 SUZ12, SUZ12C, showed that only EZH2, but not RBBP7, PHF1, or AEBP2, was 187 enriched in the GFP-Nup98N-SUZ12C compartments ( Figure S2B ). On the contrary, CoPIC assays utilizing the full-length SUZ12 showed substantial enrichment of the 189 signals of RBBP7, PHF1, or AEBP2 within the compartments of GFP-Nup98N-SUZ12 190 ( Figure S2B ). 191 Because we demonstrated that CoPIC can robustly detect direct PPIs, we conducted It is well known that ORF1ab of coronavirus is translated upon ribosomal frameshift 288 inside ORF1a, implying significantly lower levels in producing Nsp12-Nsp16 than 289 ORF1a-encoded products. Even so, Nsp12-Nsp16 are thought to serve as a platform to (Figure 3, S4B, S4C) . Notably, CoPIC allows the compartmentalization of multiple PPIs 414 simultaneously which is hard to achieve by other approaches and is important for the It is striking to identify 32 novel interactions among RTC-related factors in our study, 444 several of which were confirmed using co-IP (Figure 4, 5) . 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