RESUMO
Non-structural protein 3 (Nsp3) is the largest protein encoded by the coronavirus (CoV) genome. It consists of multiple domains that perform critical functions during the viral life cycle. CoV-Y is the most C-terminal domain of Nsp3, and it exhibits evolutionary conservation across diverse CoVs; however, the exact biological function of CoV-Y remains unclear. Here, we determined the crystal structure of CoV-Y of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Nsp3 using the single-wavelength anomalous diffraction method. We revealed the interaction between CoV-Y and the host BRCA1-associated protein (BRAP) using immunoprecipitation-mass spectrometry experiments. This interaction was subsequently confirmed in cellular assays, and the precise binding-regions between these two proteins were clarified. We found that this interaction is conserved in SARS-CoV and Middle East respiratory syndrome coronavirus. Next, we demonstrated that CoV-Y enhances IκBα and IκBß phosphorylation and promotes the nuclear translocation of the downstream NF-κB members p50 and p65 through binding to BRAP. The CoV-Y-BRAP interaction can upregulate the transcript levels of the host inflammatory cytokines. Overall, our findings illustrate the biological function of CoV-Y for the first time and provide novel insights into coronavirus regulation of host inflammatory responses, as well as a possible target for antiviral drug development.
RESUMO
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to pose a significant global health threat. Identifying new druggable targets is crucial for the advancement of drug development. Equally critical is the development of precise methods for monitoring Mtb to effectively combat this disease. Addressing these needs, our study pinpointed the pore domain (PD) of MtbMmpL3 as a new binding site for virtual screening, which led to the discovery of the small molecule ZY27. To confirm the binding site and action mode of ZY27, we employed cosolvent molecular dynamics (CMD), steered molecular dynamics (SMD), and long timescale molecular dynamics (MD) simulations of 5 µs. These in silico studies verified that ZY27 binds to the PD of MtbMmpL3. In antimicrobial activity tests, ZY27 exhibited potent anti-Mtb activity and high selectivity among mycobacterial species. Whole-genome sequencing of spontaneous ZY27-resistant Mtb variants, complemented by acid-fast staining experiments, confirmed that ZY27 specifically targets MtbMmpL3. Utilizing the ligand-protein binding data, we designed and synthesized two solvatochromic fluorescent probes, 27FP1 and 27FP2, based on ZY27. Further investigations through flow cytometry and confocal microscopy confirmed that these probes specifically label Mtb cells via the MtbMmpL3 binding mechanism.
Assuntos
Antituberculosos , Proteínas de Bactérias , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis , Mycobacterium tuberculosis/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Antituberculosos/farmacologia , Antituberculosos/química , Descoberta de Drogas/métodos , Sítios de Ligação , Ligação Proteica , Domínios Proteicos , Corantes Fluorescentes/química , Humanos , Testes de Sensibilidade Microbiana , Proteínas de Membrana TransportadorasRESUMO
The coronavirus nucleocapsid (N) protein interacts with non-structural protein 3 (Nsp3) to facilitate viral RNA synthesis and stabilization. However, structural information on the N-Nsp3 complex is limited. Here, we report a 2.6 Å crystal structure of the N-terminal domain (NTD) of the N protein in complex with the ubiquitin-like domain 1 (Ubl1) of Nsp3 in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One NTD and two Ubl1s formed a stable heterotrimer. We performed mutational analysis to reveal the key residues for this interaction. We confirmed the colocalization of SARS-CoV-2 N and Nsp3 in Huh-7 cells. N-Ubl1 interaction also exists in SARS-CoV and Middle East respiratory syndrome coronavirus. We found that SARS-CoV-2 Ubl1 competes with RNA to bind N protein in a dose-dependent manner. Based on our results, we propose a model for viral ribonucleoprotein dissociation through N protein binding to Ubl1 of Nsp3.
Assuntos
COVID-19 , Proteínas do Nucleocapsídeo , Humanos , SARS-CoV-2/genética , RNA Viral/genética , RibonucleoproteínasRESUMO
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continually poses serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing Mpro inhibitors derived from either boceprevir or telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 Mpro activity in vitro, with 50% inhibitory concentration values ranging from 7.6 to 748.5 nM. The cocrystal structure of Mpro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a transgenic mouse model of SARS-CoV-2 infection, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.