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Review
. 2023 Feb 3;12(2):243.
doi: 10.3390/pathogens12020243.

Phase Separation: The Robust Modulator of Innate Antiviral Signaling and SARS-CoV-2 Infection

Yi Zheng  1 , Chengjiang Gao  1
Affiliations
Review

Phase Separation: The Robust Modulator of Innate Antiviral Signaling and SARS-CoV-2 Infection

Yi Zheng et al. Pathogens. .

Abstract

SARS-CoV-2 has been a pandemic threat to human health and the worldwide economy, but efficient treatments are still lacking. Type I and III interferons are essential for controlling viral infection, indicating that antiviral innate immune signaling is critical for defense against viral infection. Phase separation, one of the basic molecular processes, governs multiple cellular activities, such as cancer progression, microbial infection, and signaling transduction. Notably, recent studies suggest that phase separation regulates antiviral signaling such as the RLR and cGAS-STING pathways. Moreover, proper phase separation of viral proteins is essential for viral replication and pathogenesis. These observations indicate that phase separation is a critical checkpoint for virus and host interaction. In this study, we summarize the recent advances concerning the regulation of antiviral innate immune signaling and SARS-CoV-2 infection by phase separation. Our review highlights the emerging notion that phase separation is the robust modulator of innate antiviral signaling and viral infection.

Keywords: N protein; NSP8; RLR; SARS-CoV-2; cGAS–STING; phase separation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Phase separation in the regulation of antiviral innate immune signaling. The liquid–liquid phase separation of cGAS protects DNA from the degradation of Trex1 to enhance the cGAMP production, which is promoted by G3BP1 and USP15 and antagonized by ORF52 and PCBP2. cGAMP interaction with STING leads to its trafficking from ER to Golgi for downstream TBK1 activation, but part of cGAMP also induces the gel-like droplets of STING, which includes unphosphorylated TBK1 on the ER membranes. Stress granules driven by the LLPS of G3BP1 include RIG-I and MDA5. The activation of RIG-I and MDA5 further stimulates the aggregation of MAVS on the mitochondria in liquid-like droplets. cGAS–STING signaling and RLR-MAVS signaling are converged on the kinase TBK1 and transcriptional factor IRF3. Phosphorylated and dimerized IRF3 translocates into the nucleus and forms condensates with the ISRE DNA element to drive the type I interferon response.
Figure 2
Figure 2
The domain structure of the phase-separated proteins discussed in this study and the domains required for their phase separation. (A) The domain structure of cGAS. cGAS is composed of the N-terminal IDR, middle NTase core, and C-terminal Mab21 domain. The N-terminal IDR region is required for its phase separation. The NTase core (residues 161–330) is the core enzymatic part for the synthesis of cGAMP. Residues from 389 to 405 represent a zinc-ribbon domain for binding with zinc, which stabilizes the DNA-binding and cGAS dimers. (B) The domain structure of STING. STING is composed of a cytosolic N-terminal tail, N-terminal four transmembrane (TM) domains, dimerization domain (DD), ligand-binding domain (LBD), and C-terminal tail (CTT). The IDR region (residues 309–343) is located within the LBD domain. TM, DD, and IDR are critical for the phase separation of STING. Within IDR, E336/E337 are the two essential residues for phase separation. (C) The domain structure of IRF3 and IRF7. IRF3 is composed of a DNA-binding domain (DBD), IDR region, IRF-associated domain (IAD) for dimerization, and signal response domain (SRD) containing serine residues 386 and 396 for activation. DBD, IDR, and IAD domains are required for its phase separation, while the SRD domain plays a negative role in its LLPS. IRF7 is composed of a DNA-binding domain (DBD), IDR region, IRF-associated domain (IAD) for dimerization, and signal response domain (SRD). DBD, IDR, and IAD domains are required for its phase separation, while the SRD domain plays a negative role in its LLPS. (D) The domain structure of the N protein of SARS-CoV-2. The N protein is composed of the N arm (NIDR), the N-terminal domain (NTD), the linker region (LinkerIDR or S/R rich motif), the C-terminal domain (CTD), and the C-terminal tail (CIDR). NTD and CTD bind with RNA, and CTD is the dimerization domain. NIDR interacts with G3BP1, and LinkerIDR binds with NSP3. Within the LinkerIDR or S/R rich motif, the phosphorylation is important for the N protein to be involved in the viral transcription and replication. LinkerIDR and the CTD domains are essential for its phase separation. (E) The domain structure of the NSP8 protein of SARS-CoV-2. The NSP8 protein is composed of an N-terminal extension region (IDR: residues 1–76) and a C-terminal head region (interaction with NSP7 and NSP12). The N-terminal region is positively charged and responsible for binding with single-stranded nucleic acids and interaction with helicase NSP13. The C-terminal head region is crucial for binding with the NSP7 and NSP12 proteins to form the holo–RdRP complex. The N-terminal IDR region is required for its LLPS.
Figure 3
Figure 3
Functions of N protein phase separation. Phase separation of the N protein promotes viral genome packaging/virion assembly, RTC formation, and virus-induced inflammation. Phase separation of the N protein counteracts the RLR-MAVS mediated type I interferon response. Phase separation of N promotes viral genome packaging/virion assembly with the interaction between N and viral genomic RNA as well as the interaction between the N protein and M protein intravirion tail (left panel). RTC formation is driven by the N condensates with NSP3, NSP12, NSP7, and NSP8 (middle left panel). The phosphorylation of the N protein plays a critical switch between these two functions, with unphosphorylated N protein forming gel-like condensate to promote viral genome packaging and phosphorylated N protein facilitating viral replication and transcription. N protein condensates harboring TAK1 and IKK also facilitate inflammation (middle right panel). N protein condensates prevent the SG formation and aggregation of MAVS to inhibit the type I interferon response (right panel).
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