Compartmentalization-aided interaction screening reveals extensive high-order complexes within the SARS-CoV-2 proteome

Abstract

Bearing a relatively large single-stranded RNA genome in nature, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes sophisticated replication/transcription complexes (RTCs), mainly composed of a network of nonstructural proteins and nucleocapsid protein, to establish efficient infection. In this study, we develop an innovative interaction screening strategy based on phase separation in cellulo, namely compartmentalization of protein-protein interactions in cells (CoPIC). Utilizing CoPIC screening, we map the interaction network among RTC-related viral proteins. We identify a total of 47 binary interactions among 14 proteins governing replication, discontinuous transcription, and translation of coronaviruses. Further exploration via CoPIC leads 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 they indicate the existence of a complex interaction network among RTC-related factors, thus opening up opportunities to understand SARS-CoV-2 biology and develop therapeutic interventions for COVID-19.

Keywords: CoPIC; PRC2; RTC; SARS-CoV-2; high-order complexes; nonstructural proteins; protein-protein interaction.

Graphical abstract

Figure 1

Establishment of a robust PPI-mediated recruitment system in cells (A) Schematic diagram showing the strategy of CoPIC. The direct interaction between the protein of interest (POI) and the potential binding client is assessed by the enrichment of mCherry signals into the phase-separated compartments of the GFP-labeled scaffold. (B) The domain structures of scaffold candidates tested in CoPIC. (C) Characterization of scaffold candidates fused with GFP in HEK293 cells, with the nucleus stained by DAPI. Scale bar, 5 μm. All assays were performed in triplicate. (D and F) Validation of the direct interaction between GFP-Nup98N-MDM2 and mCherry-p53 using CoPIC. The p53 fusion protein, as indicated by the mCherry signal, was recruited to the green compartment of GFP-Nup98N-MDM2 by the specific interaction (D). The co-expression of GFP-Nup98N and mCherry-p53 served as the control (F). Scale bars, 5 μm. All assays were performed in triplicate. (E and G) Fluorescent intensity profiles of the lines with white arrows from (D) and (F). (H) Treatment of cells co-expressing GFP-Nup98N-MDM2 and mCherry-p53 with Mi-773, an inhibitor of the MDM2/p53 interaction. Scale bar, 5 μm. All assays were performed in triplicate. (I) Plot of the intensity ratio of mCherry versus GFP under Mi-773 treatment. Each data point was determined by three independent assays, and error bars represent standard deviations.

Figure 2

Characterization of direct and indirect interactions within the PRC2 complex using CoPIC (A) CoPIC analysis of the positive interaction between SUZ12 and RBBP4. Scale bar, 5 μm. All assays were performed in triplicate. (C) CoPIC analysis of the negative interaction between EZH2 and RBBP7. Scale bar, 5 μm. All assays were performed in triplicate. (E) Summary of all pairwise interactions between PRC2 core subunits, including EZH1/2, SUZ12, EED, and RBBP4/7. (F) Domain structures of SUZ12 and SUZ12C (C-terminal region of SUZ12, known to bind to EZH2 but not RBBP7). (G) Schematic diagram showing the known factors binding to SUZ12. (H) CoPIC analysis of indirect interaction of EZH2 with RBBP7 mediated by SUZ12. Scale bar, 5 μm. All assays were performed in triplicate. (J) Verification of the bridging role of full-length SUZ12 but not SUZ12C in recruiting RBBP7 into EZH2-containing compartments. Scale bar, 5 μm. All assays were performed in triplicate. (B, D, I, and K) Fluorescence intensity profiles of the lines with white arrows in (A), (C), (H), and (J), respectively, are shown.

Figure 3

CoPIC screening of intraviral PPIs within SARS-CoV-2 RTC-related factors (A) Summary of intraviral interactions between selected factors associated with RTCs. The grids with black/blue/red fill represent unidirectional/bidirectional/self-interactions identified by CoPIC while the gray ones are negative interactions. (B) Analysis of the proportion of each type of interaction for all pairwise interactions. (C) Annotation for the interaction network with degree-sorted layout.

Figure 4

Analysis of interaction patterns between SARS-CoV and SARS-CoV-2 (A) Comparison of the intraviral interactions of SARS-CoV-2 (detected by CoPIC) with SARS-CoV (reported in the literature). The grids with magenta fill represent the positive interactions identified both in SARS-CoV and SARS-CoV-2; green indicates positive interactions in SARS-CoV while negative in CoPIC screening of SARS-CoV-2; cyan indicates the interactions only in SARS-CoV-2 identified by CoPIC; and gray indicates negative interactions both in SARS-CoV and SARS-CoV-2. (B, D, F, and H) CoPIC analyses of the representative pairwise PPIs, in which Nsp5-Nsp8 is the negative interaction in SARS-CoV-2 identified by CoPIC, Nsp5-Nsp12 is a well-known interaction both in SARS-CoV and SARS-CoV-2, Nsp5-Nsp9 is an interaction in SARS-CoV-2 identified by CoPIC, and Nsp9-Nsp9 is a self-interaction. Scale bar, 5 μm. All assays were performed in triplicate. (C, E, G, and I) Fluorescence intensity profiles of the lines with white arrows are shown. (J–M) Coimmunoprecipitation analyses of the representative pairwise PPIs (B, D, F, and H).

Figure 5

Mapping of extensive high-order interactions within SARS-CoV-2 (A) Network of selected viral proteins as the blueprint for mapping high-order complexes. The black solid lines are shown as the direct binary interactions while the red dashed lines are potential indirect interactions with the aid of extra intermediates. The triangles are the hypothetical or CoPIC-verified tertiary complexes and are listed in the outer ring and middle ring, respectively. (B and C) Interpretation of the patterns of high-order complexes. Direct contacts of A with both B and C mediate the indirect interaction of B with C, thus forming tertiary complexes shown in the middle ring (B). On the contrary, the mode in the outer ring illustrates two possibilities (C): (1) a potential tertiary complex, in which all of the components can interact with each other, and (2) the combinations of multiple binary interactions.

Figure 6

Characterization of representative indirect interactions of Nsp7-Nsp8-Nsp16 and Nsp10-N-Nsp13 (A) CoPIC analysis of the pairwise interactions within the positive combinations of Nsp7-Nsp8-Nsp16. Scale bars, 5 μm. All assays were performed in triplicate. (B) Fluorescent intensity profiles of the lines with white arrows from (A). (C) CoPIC analysis of the pairwise interactions within the negative combinations of Nsp10-N-Nsp13. Scale bars, 5 μm. All assays were performed in triplicate.

Figure 7

The outlook for higher-order complexes and a potential HTS scheme for PPI inhibitors (A–C) Simplified model systems deciphering the potential higher-order complexes. (D) Schematic diagram of high-throughput screening for specific inhibitors against intraviral complexes.