Molecular Mechanisms and Potential Antiviral Strategies of Liquid-Liquid Phase Separation during Coronavirus Infection

Abstract

Highly pathogenic coronaviruses have caused significant outbreaks in humans and animals, posing a serious threat to public health. The rapid global spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in millions of infections and deaths. However, the mechanisms through which coronaviruses evade a host's antiviral immune system are not well understood. Liquid-liquid phase separation (LLPS) is a recently discovered mechanism that can selectively isolate cellular components to regulate biological processes, including host antiviral innate immune signal transduction pathways. This review focuses on the mechanism of coronavirus-induced LLPS and strategies for utilizing LLPS to evade the host antiviral innate immune response, along with potential antiviral therapeutic drugs and methods. It aims to provide a more comprehensive understanding and novel insights for researchers studying LLPS induced by pandemic viruses.

Keywords: SARS-CoV-2; innate immunity; liquid–liquid phase separation; nucleocapsid protein.

Figure 1

The classes of Coronavirus and their life cycle. Coronaviruses are classified into four distinct genera: α-CoV, β-CoV, γ-CoV, and δ-CoV. The life cycle of the four genera of coronaviruses is essentially similar, except for differences in entry into the cellular receptor (e.g., SARS-CoV-2 spiny protein binding to ACE2, TGEV and PDCoV binding to porcine APN). Coronavirus spiking proteins bind to the cellular receptor and enter the intracellular space by plasma membrane fusion in the presence of cytosolic proteases. Viral N proteins and RNA are released, and the ORF1a/b proteins are translated and processed into non-structural proteins to form RTCs and DMVs, with the RNA replicating in the DMVs and eventually extruding from the membrane pores. Structural proteins are translated and then transported to the Golgi apparatus via ERGIC. These newly synthesized viral structural proteins (N, E, M, and S) and genomic +ssRNA are reassembled to form progeny virus, which eventually exits the cell.

Figure 2

LLPS in the innate immune pathway. After stimulation by viral RNA/DNA, key immune molecules such as RIG-I, MAVS, cGAS, IRF3, and STING undergo LLPS to activate IFN signaling. Positive regulation of antiviral response is also present with TRIM25, NEMO, TFAM, HMGB, etc. Antiviral factors such as TRIM5a, MX1, NLRP3, NLRP6, etc., form liquid-like condensates and mediate antiviral immune response. At the same time, non-structural proteins of coronaviruses such as Nsp5 and Nsp1 participate in the negative regulation of antiviral responses.

Figure 3

The conserved domains of different CoV-encoded N proteins are included, NTD and CTD, and the LKR. In particular, SARS-CoV-2 N protein contains a special serine-rich (SR) region, which has large differences among different coronavirus genera. Moreover, SARS-CoV-2 N protein contains three intrinsically disordered regions (IDRs), including an N-terminal IDR, a center IDR, and a C-terminal IDR.

Figure 4

Summary of LLPS-based therapeutic strategies. (a). Small-molecule oligo-gua nosine RNA affects LLPS: G12 inhibits liquid-phase separation by expelling RNA from separated droplets, and G6 forms unstable liquid-phase structures. (b). Competition for ATP binding to the RBD and expulsion of RNA from the liquid-phase structure inhibits LLPS. (c). Screening of genes regulating G3BP1/2 resulted in inhibition of IFN induction by screening for imatinib and decitabine to inhibit LLPS by taking advantage of their much higher binding to G3BP1/2 than the coronavirus N protein property to inhibit IFN induction. (d). GCG selectively binds N proteins to inhibit LLPS.