Mechanisms and targets in SARS-CoV-2 : from viral protein interactions to antiviral strategies

SARS-COV-2 is an enveloped, positive-stranded RNA virus belonging to the ß- coronaviruses. The interplay between viral proteins and host factors is critical for viral propagation and immune evasion. Extensive virus-host interactome studies have revealed numerous host proteins that modulate SARS-CoV-2 replication, including the interaction between G3BP and the viral N protein.

G3BP1, a key RNA-binding protein, is central to assembling SGs in response to various stressors, including viral infection. In Paper I, we demonstrate that the SARS-CoV-2 transiently induced SGs formation at early infection, then N protein binds to G3BP via a conserved ITFG motif within its intrinsically disordered region (N-IDR1), which triggers SG disassembly despite persistent PKR-elF2a activation. An engineered RATA virus lacking this G3BP-binding motif in the N protein induces sustained SG formation, resulting in reduced viral replication in vitro and complete attenuation of pathogenicity in K18-hACE2 mice, highlighting the importance of G3BP-N interaction in SARS-CoV-2 infection. Our data further reveal that N protein recruits G3BP1 to viral replication-transcription complex (RTC) through nsp3 scaffolding at double-membrane vesicle (DMV) pores during the early stage of infection; subsequently, G3BPI's RGG domain concentrates 40S ribosomes to facilitate localized viral mRNA translation. Collectively, these findings illustrate how SARS-CoV-2 strategically exploits G3BP1 to evade antiviral stress responses and enhance replication, offering promising avenues for therapeutic intervention.

In Paper II, we employed drug repurposing to screen for antiviral agents by targeting conserved nucleotide-binding pockets (NBPs) across six viral proteins (nsp12, nsp13, nsp14, nsp15, nsp16, and N protein). These NBPs are evolutionarily constrained due to their essential roles in viral replication. Through structure- based screening of three compound libraries-the FDA-approved drug library, the natural product library (NPL), and the LOPAC library-we identified several promising candidates. Subsequent validation using isothermal titration calorimetry (ITC) and in vitro antiviral assays confirmed INCB28060, darglitazone sodium, and columbianadin as potent inhibitors of SARS-CoV-2 replication.

In summary, Paper I clarifies a key host-pathogen interaction by demonstrating the complex role of the G3BP-N interaction in viral replication, thereby providing a conserved target for antiviral therapy; Paper II targets conserved viral features NBPs for therapeutic development, addressing both viral evasion mechanisms and clinical applicability.

List of scientific papers

This thesis is based on the following publications

I. SARS-CoV-2 N protein recruits G3BP to double membrane vesicles to promote translation of viral mRNAs. Long S; Guzyk M; Vidakovics LP; Han X; Sun R; Wang M; Panas MD; Urgard E; Coquet JM; Merits A; Achour A; Mcinerney GM. NATURE COMMUNICATIONS. 2024;15(1):10607. DOI: https://doi.org/10.1038/s41467-024-54996-3

II. Multi-target direct-acting SARS-CoV-2 antivirals against the nucleotide- binding pockets of virus-specific proteins. Rani R; Long S; Pareek A; Dhaka P; Singh A; Kumar P; McInerney G; Tomar S. VIROLOGY. 2022;577:1-15. DOI: https://doi.org/10.1016/j.virol.2022.08.008