RESUMO
The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.
Assuntos
Proteínas do Nucleocapsídeo de Coronavírus , SARS-CoV-2 , COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Humanos , Microscopia Eletrônica , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , RNA Viral/genética , SARS-CoV-2/química , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMO
The outbreak of COVID-19, the disease caused by SARS-CoV-2, continues to affect millions of people around the world. The absence of a globally distributed effective treatment makes the exploration of new mechanisms of action a key step to address this situation. Stabilization of non-native Protein-Protein Interactions (PPIs) of the nucleocapsid protein of MERS-CoV has been reported as a valid strategy to inhibit viral replication. In this study, the applicability of this unexplored mechanism of action against SARS-CoV-2 is analyzed. During our research, we were able to find three inducible interfaces of SARS-CoV-2 N protein NTD, compare them to the previously reported MERS-CoV stabilized dimers, and identify those residues that are responsible for their formation. A drug discovery protocol implemented consisting of docking, molecular dynamics and MM-GBSA enabled us to find several compounds that might be able to exploit this mechanism of action. In addition, a common catechin skeleton was found among many of these molecules, which might be useful for further drug design. We consider that our findings could motivate future research in the fields of drug discovery and design towards the exploitation of this previously unexplored mechanism of action against COVID-19.Communicated by Ramaswamy H. Sarma.
Assuntos
Tratamento Farmacológico da COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus , Descoberta de Drogas , Proteínas do Nucleocapsídeo de Coronavírus/antagonistas & inibidores , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Inibidores de Proteases , Mapeamento de Interação de Proteínas , SARS-CoV-2/efeitos dos fármacos , Replicação ViralRESUMO
The SARS-CoV-2 N protein binds several cell host proteins including 14-3-3γ, a well-characterized regulatory protein. However, the biological function of this interaction is not completely understood. We analyzed the variability of â¼90 000 sequences of the SARS-CoV-2 N protein, particularly, its mutations in disordered regions containing binding motifs for 14-3-3 proteins. We studied how these mutations affect the binding energy to 14-3-3γ and found that changes positively affecting the predicted interaction with 14-3-3γ are the most successfully spread, with the highest prevalence in the phylogenetic tree. Although most residues are highly conserved within the 14-3-3 binding site, compensatory mutations to maintain the interaction energy of N-14-3-3γ were found, including half of the current variants of concern and interest. Our results suggest that binding of N to 14-3-3γ is beneficial for the virus, thus targeting this viral-host protein-protein interaction seems an attractive approach to explore antiviral strategies.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/análise , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Sítios de Ligação , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Mutação/genética , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Filogenia , Ligação ProteicaRESUMO
In the current global emergency due to SARS-CoV-2 outbreak, passive immunotherapy emerges as a promising treatment for COVID-19. Among animal-derived products, equine formulations are still the cornerstone therapy for treating envenomations due to animal bites and stings. Therefore, drawing upon decades of experience in manufacturing snake antivenom, we developed and preclinically evaluated two anti-SARS-CoV-2 polyclonal equine formulations as potential alternative therapy for COVID-19. We immunized two groups of horses with either S1 (anti-S1) or a mixture of S1, N, and SEM mosaic (anti-Mix) viral recombinant proteins. Horses reached a maximum anti-viral antibody level at 7 weeks following priming, and showed no major adverse acute or chronic clinical alterations. Two whole-IgG formulations were prepared via hyperimmune plasma precipitation with caprylic acid and then formulated for parenteral use. Both preparations had similar physicochemical and microbiological quality and showed ELISA immunoreactivity towards S1 protein and the receptor binding domain (RBD). The anti-Mix formulation also presented immunoreactivity against N protein. Due to high anti-S1 and anti-RBD antibody content, final products exhibited high in vitro neutralizing capacity of SARS-CoV-2 infection, 80 times higher than a pool of human convalescent plasma. Pre-clinical quality profiles were similar among both products, but clinical efficacy and safety must be tested in clinical trials. The technological strategy we describe here can be adapted by other producers, particularly in low- and middle-income countries.