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

<p dir="ltr">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.</p><p dir="ltr">G3BP1, a key RNA-binding protein, is central to assembling SGs in response to various stressors, including viral infection. <b>In Paper I</b>, 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.</p><p dir="ltr"><b>In Paper II</b>, 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.</p><p dir="ltr">In summary, <b>Paper I</b> 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; <b>Paper II </b>targets conserved viral features NBPs for therapeutic development, addressing both viral evasion mechanisms and clinical applicability.</p><h3>List of scientific papers</h3><p dir="ltr">This thesis is based on the following publications</p><p dir="ltr">I. SARS-CoV-2 N protein recruits G3BP to double membrane vesicles to promote translation of viral mRNAs. <b>Long S;</b> 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: <a href="https://doi.org/10.1038/s41467-024-54996-3" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41467-024-54996-3</a></p><p dir="ltr">II. Multi-target direct-acting SARS-CoV-2 antivirals against the nucleotide- binding pockets of virus-specific proteins. Rani R; <b>Long S;</b> Pareek A; Dhaka P; Singh A; Kumar P; McInerney G; Tomar S. VIROLOGY. 2022;577:1-15. DOI: <a href="https://doi.org/10.1016/j.virol.2022.08.008" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.virol.2022.08.008</a></p>