RESUMEN
The nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 encapsidates the viral genome and is essential for viral function. The central disordered domain comprises a serine-arginine-rich (SR) region that is hyperphosphorylated in infected cells. This modification regulates function, although mechanistic details remain unknown. We use nuclear magnetic resonance to follow structural changes occurring during hyperphosphorylation by serine arginine protein kinase 1, glycogen synthase kinase 3, and casein kinase 1, that abolishes interaction with RNA. When eight approximately uniformly distributed sites have been phosphorylated, the SR domain binds the same interface as single-stranded RNA, resulting in complete inhibition of RNA binding. Phosphorylation by protein kinase A does not prevent RNA binding, indicating that the pattern resulting from physiologically relevant kinases is specific for inhibition. Long-range contacts between the RNA binding, linker, and dimerization domains are abrogated, phenomena possibly related to genome packaging and unpackaging. This study provides insight into the recruitment of specific host kinases to regulate viral function.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus , Unión Proteica , ARN Viral , SARS-CoV-2 , Fosforilación , SARS-CoV-2/metabolismo , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/química , Humanos , ARN Viral/metabolismo , ARN Viral/química , Conformación Proteica , COVID-19/virología , COVID-19/metabolismo , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/química , Modelos Moleculares , Sitios de Unión , FosfoproteínasRESUMEN
The spread of the SARS-CoV-2 virus has had an unprecedented impact, both by posing a serious risk to human health and by amplifying the burden on the global economy. The rapid identification of the SARS-CoV-2 virus has been crucial to preventing and controlling the spread of SARS-CoV-2 infections. In this study, we propose a multilayered plasmonic nanotrap (MPNT) device for the rapid identification of single particles of SARS-CoV-2 virus in ultra-high sensitivity by surface-enhanced Raman scattering (SERS). The MPNT device is composed of arrays of concentric cylindrical cavities with Ag/SiO2/Ag multilayers deposited on the top and at the bottom. By varying the diameter of the cylinders and the thickness of the multilayers, the resonant optical absorption and local electric field were optimized. The SERS enhancement factors of the proposed device are of the order of 108, which enable the rapid identification of SARS-CoV-2 N protein in concentrations as low as 1.25 × 10-15ï¼12.5 × 10-15 g mL-1 within 1 min. The developed MPNT SERS device provides a label-free and rapid detection platform for SARS-CoV-2 virus. The general nature of the device makes it equally suitable to detect other infectious viruses.
Asunto(s)
COVID-19 , SARS-CoV-2 , Plata , Espectrometría Raman , Espectrometría Raman/métodos , SARS-CoV-2/aislamiento & purificación , Plata/química , Humanos , COVID-19/diagnóstico , COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/análisis , Dióxido de Silicio/química , Fosfoproteínas/análisis , Fosfoproteínas/química , Nanopartículas del Metal/química , Límite de Detección , Proteínas de la Nucleocápside/químicaRESUMEN
Puumala orthohantavirus (PUUV) is an emerging zoonotic virus endemic to Europe and Russia that causes nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome (HFRS). There are limited options for treatment and diagnosis of orthohantavirus infection, making the search for potential immunogenic candidates crucial. In the present work, various bioinformatics tools were employed to design conserved immunogenic peptides containing multiple epitopes of PUUV nucleocapsid protein. Eleven conserved peptides (90% conservancy) of the PUUV nucleocapsid protein were identified. Three conserved peptides containing multiple T and B cell epitopes were selected using a consensus epitope prediction algorithm. Molecular docking using the HPEP dock server demonstrated strong binding interactions between the epitopes and HLA molecules (ten alleles for each class I and II HLA). Moreover, an analysis of population coverage using the IEDB database revealed that the identified peptides have over 90% average population coverage across six continents. Molecular docking and simulation analysis reveal a stable interaction with peptide constructs of chosen immunogenic peptides and Toll-like receptor-4. These computational analyses demonstrate selected peptides' immunogenic potential, which needs to be validated in different experimental systems.
Asunto(s)
Simulación del Acoplamiento Molecular , Proteínas de la Nucleocápside , Péptidos , Virus Puumala , Virus Puumala/inmunología , Virus Puumala/genética , Péptidos/inmunología , Péptidos/química , Humanos , Proteínas de la Nucleocápside/inmunología , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/química , Fiebre Hemorrágica con Síndrome Renal/inmunología , Fiebre Hemorrágica con Síndrome Renal/virología , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/química , Biología Computacional , Secuencia Conservada , Secuencia de Aminoácidos , Unión ProteicaRESUMEN
Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also observe functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.
Like other types of RNA viruses, the genetic material of SARS-CoV-2 (the agent responsible for COVID-19) is formed of an RNA molecule which is prone to accumulating mutations. This gives SARS-CoV-2 the ability to evolve quickly, and often to remain one step ahead of treatments. Understanding how these mutations shape the behavior of RNA viruses is therefore crucial to keep diseases such as COVID-19 under control. The gene that codes for the protein that 'packages' the genetic information inside SARS-CoV-2 is particularly prone to mutations. This nucleocapsid (N) protein participates in many key processes during the life cycle of the virus, including potentially interfering with the immune response. Exactly how the physical properties of the N-Protein are impacted by the mutations in its genetic sequence remains unclear. To investigate this question, Nguyen et al. predicted the various biophysical properties of different regions of the N-protein based on a computer-based analysis of SARS-CoV-2 genetic databases. This allowed them to determine if specific protein regions were positively or negatively charged in different mutants. The analyses showed that some domains exhibited great variability in their charge between protein variants reflecting the fact that the corresponding genetic sequences showed high levels of plasticity. Other regions remained conserved, however, including across related coronaviruses. Nguyen et al. also conducted biochemical experiments on a range of N-proteins obtained from clinically relevant SARS-CoV-2 variants. Their results highlighted the importance of protein segments with no fixed three-dimensional structure. Mutations in the related sequences created high levels of variation in the physical properties of these 'intrinsically disordered' regions, which had wide-ranging consequences. Some of these genetic changes even gave individual N-proteins the ability to interact with each other in a completely new way. These results shed new light on the relationship between genetic mutations and the variable physical properties of RNA virus proteins. Nguyen et al. hope that this knowledge will eventually help to develop more effective treatments for viral infections.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus , Mutación , SARS-CoV-2 , SARS-CoV-2/genética , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/metabolismo , COVID-19/virología , COVID-19/genética , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/química , Termodinámica , Estabilidad ProteicaRESUMEN
The Nucleocapsid (N) protein of SARS-CoV-2 plays a crucial role in viral replication and pathogenesis, making it an attractive target for developing antiviral therapeutics. In this study, we used differential scanning fluorimetry to establish a high-throughput screening method for identifying high-affinity ligands of N-terminal domain of the N protein (N-NTD). We screened an FDA-approved drug library of 1813 compounds and identified 102 compounds interacting with N-NTD. The screened compounds were further investigated for their ability to inhibit the nucleic-acid binding activity of the N protein using electrophoretic mobility-shift assays. We have identified three inhibitors, Ceftazidime, Sennoside A, and Tannic acid, that disrupt the N protein's interaction with RNA probe. Ceftazidime and Sennoside A exhibited nano-molar range binding affinities with N protein, determined through surface plasmon resonance. The binding sites of Ceftazidime and Sennoside A were investigated using [1H, 15N]-heteronuclear single quantum coherence (HSQC) NMR spectroscopy. Ceftazidime and Sennoside A bind to the putative RNA binding site of the N protein, thus providing insights into the inhibitory mechanism of these compounds. These findings will contribute to the development of novel antiviral agents targeting the N protein of SARS-CoV-2.
Asunto(s)
Antivirales , Proteínas de la Nucleocápside de Coronavirus , SARS-CoV-2 , Antivirales/farmacología , Antivirales/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/antagonistas & inhibidores , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Sitios de Unión , Humanos , Unión Proteica , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/antagonistas & inhibidores , Taninos/química , Taninos/farmacología , Tratamiento Farmacológico de COVID-19 , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/antagonistas & inhibidores , Proteínas de la Nucleocápside/metabolismoRESUMEN
We report the first cryoEM structure of the Hendra henipavirus nucleoprotein in complex with RNA, at 3.5 Å resolution, derived from single particle analysis of a double homotetradecameric RNA-bound N protein ring assembly exhibiting D14 symmetry. The structure of the HeV N protein adopts the common bi-lobed paramyxoviral N protein fold; the N-terminal and C-terminal globular domains are bisected by an RNA binding cleft containing six RNA nucleotides and are flanked by the N-terminal and C-terminal arms, respectively. In common with other paramyxoviral nucleocapsids, the lateral interface between adjacent Ni and Ni+1 protomers involves electrostatic and hydrophobic interactions mediated primarily through the N-terminal arm and globular domains with minor contribution from the C-terminal arm. However, the HeV N multimeric assembly uniquely identifies an additional protomer-protomer contact between the Ni+1 N-terminus and Ni-1 C-terminal arm linker. The model presented here broadens the understanding of RNA-bound paramyxoviral nucleocapsid architectures and provides a platform for further insight into the molecular biology of HeV, as well as the development of antiviral interventions.
Asunto(s)
Microscopía por Crioelectrón , Virus Hendra , Nucleocápside , Nucleoproteínas , Virus Hendra/química , Nucleoproteínas/química , Nucleoproteínas/ultraestructura , Nucleoproteínas/metabolismo , Nucleocápside/química , Nucleocápside/ultraestructura , Nucleocápside/metabolismo , Modelos Moleculares , ARN Viral/química , ARN Viral/metabolismo , ARN Viral/genética , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/ultraestructura , Proteínas de la Nucleocápside/metabolismoRESUMEN
Influenza poses a substantial health risk, with infants and the elderly being particularly susceptible to its grave impacts. The primary challenge lies in its rapid genetic evolution, leading to the emergence of new Influenza A strains annually. These changes involve punctual mutations predominantly affecting the two main glycoproteins: Hemagglutinin (HA) and Neuraminidase (NA). Our existing vaccines target these proteins, providing short-term protection, but fall short when unexpected pandemics strike. Delving deeper into Influenza's genetic makeup, we spotlight the nucleoprotein (NP) - a key player in the transcription, replication, and packaging of RNA. An intriguing characteristic of the NP is that it is highly conserved across all Influenza A variants, potentially paving the way for a more versatile and broadly protective vaccine. We designed and synthesized a novel NP-Hoc fusion protein combining Influenza A nucleoprotein and T4 phage Hoc, cloned using Gibson assembly in E. coli, and purified via ion affinity chromatography. Simultaneously, we explore the T4 coat protein Hoc, typically regarded as inconsequential in controlled viral replication. Yet, it possesses a unique ability: it can link with another protein, showcasing it on the T4 phage coat. Fusing these concepts, our study designs, expresses, and purifies a novel fusion protein named NP-Hoc. We propose this protein as the basis for a new generation of vaccines, engineered to guard broadly against Influenza A. The excitement lies not just in the immediate application, but the promise this holds for future pandemic resilience, with NP-Hoc marking a significant leap in adaptive, broad-spectrum influenza prevention.
Asunto(s)
Bacteriófago T4 , Escherichia coli , Proteínas Recombinantes de Fusión , Bacteriófago T4/genética , Bacteriófago T4/química , Bacteriófago T4/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/biosíntesis , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/química , Humanos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificaciónRESUMEN
The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently folded monomeric N-terminal domain that is the primary site for RNA binding and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation, we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino-acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA-binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA-binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid-liquid phase separation of the entire protein.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus , SARS-CoV-2 , Arginina/química , Arginina/metabolismo , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/genética , COVID-19/virología , COVID-19/metabolismo , Espectroscopía de Resonancia Magnética , Nucleocápside/metabolismo , Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/química , Separación de Fases , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilación , Unión Proteica , ARN Viral/metabolismo , ARN Viral/química , ARN Viral/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/química , Serina/metabolismo , Serina/químicaRESUMEN
The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus , Multimerización de Proteína , ARN Viral , SARS-CoV-2 , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/química , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/genética , ARN Viral/metabolismo , ARN Viral/química , ARN Viral/genética , Unión Proteica , Sitios de Unión , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Ensamble de Virus/genética , Humanos , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/genética , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , COVID-19/virologíaRESUMEN
The coordinated packaging of the segmented genome of the influenza A virus (IAV) into virions is an essential step of the viral life cycle. This process is controlled by the interaction of packaging signals present in all eight viral RNA (vRNA) segments and the viral nucleoprotein (NP), which binds vRNA via a positively charged binding groove. However, mechanistic models of how the packaging signals and NP work together to coordinate genome packaging are missing. Here, we studied genome packaging in influenza A/SC35M virus mutants that carry mutated packaging signals as well as specific amino acid substitutions at the highly conserved lysine (K) residues 184 and 229 in the RNA-binding groove of NP. Because these lysines are acetylated and thus neutrally charged in infected host cells, we replaced them with glutamine to mimic the acetylated, neutrally charged state or arginine to mimic the non-acetylated, positively charged state. Our analysis shows that the coordinated packaging of eight vRNAs is influenced by (i) the charge state of the replacing amino acid and (ii) its location within the RNA-binding groove. Accordingly, we propose that lysine acetylation induces different charge states within the RNA-binding groove of NP, thereby supporting the activity of specific packaging signals during coordinated genome packaging. IMPORTANCE: Influenza A viruses (IAVs) have a segmented viral RNA (vRNA) genome encapsidated by multiple copies of the viral nucleoprotein (NP) and organized into eight distinct viral ribonucleoprotein complexes. Although genome segmentation contributes significantly to viral evolution and adaptation, it requires a highly sophisticated genome-packaging mechanism. How eight distinct genome complexes are incorporated into the virion is poorly understood, but previous research suggests an essential role for both vRNA packaging signals and highly conserved NP amino acids. By demonstrating that the packaging process is controlled by charge-dependent interactions of highly conserved lysine residues in NP and vRNA packaging signals, our study provides new insights into the sophisticated packaging mechanism of IAVs.
Asunto(s)
Virus de la Influenza A , Proteínas de la Nucleocápside , Empaquetamiento del Genoma Viral , Animales , Perros , Humanos , Sustitución de Aminoácidos , Línea Celular , Genoma Viral , Virus de la Influenza A/química , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Lisina/genética , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , ARN Viral/metabolismo , Empaquetamiento del Genoma Viral/genética , Virión/química , Virión/genética , Virión/metabolismo , Mutación , Electricidad EstáticaRESUMEN
Phenuiviridae nucleoprotein is the main structural and functional component of the viral cycle, protecting the viral RNA and mediating the essential replication/transcription processes. The nucleoprotein (N) binds the RNA using its globular core and polymerizes through the N-terminus, which is presented as a highly flexible arm, as demonstrated in this article. The nucleoprotein exists in an `open' or a `closed' conformation. In the case of the closed conformation the flexible N-terminal arm folds over the RNA-binding cleft, preventing RNA adsorption. In the open conformation the arm is extended in such a way that both RNA adsorption and N polymerization are possible. In this article, single-crystal X-ray diffraction and small-angle X-ray scattering were used to study the N protein of Toscana virus complexed with a single-chain camelid antibody (VHH) and it is shown that in the presence of the antibody the nucleoprotein is unable to achieve a functional assembly to form a ribonucleoprotein complex.
Asunto(s)
Nucleoproteínas , Virus de Nápoles de la Fiebre de la Mosca de los Arenales , Nucleoproteínas/química , Virus de Nápoles de la Fiebre de la Mosca de los Arenales/genética , Virus de Nápoles de la Fiebre de la Mosca de los Arenales/metabolismo , Proteínas de la Nucleocápside/química , Modelos Moleculares , ARN Viral/química , ARN Viral/metabolismoRESUMEN
The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA binding domain (RBD) and its flanking disordered N-terminal domain (NTD) tail, using single-molecule Förster Resonance Energy Transfer and coarse-grained simulations. We quantified contact site size and binding affinity for nucleic acids and concomitant conformational changes occurring in the disordered region. We found that the disordered NTD increases the affinity of the RBD for RNA by about 50-fold. Binding of both nonspecific and specific RNA results in a modulation of the tail configurations, which respond in an RNA length-dependent manner. Not only does the disordered NTD increase affinity for RNA, but mutations that occur in the Omicron variant modulate the interactions, indicating a functional role of the disordered tail. Finally, we found that the NTD-RBD preferentially interacts with single-stranded RNA and that the resulting protein:RNA complexes are flexible and dynamic. We speculate that this mechanism of interaction enables the Nucleocapsid protein to search the viral genome for and bind to high-affinity motifs.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus , ARN Viral , SARS-CoV-2 , Humanos , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/metabolismo , COVID-19/virología , Proteínas de la Nucleocápside/química , Unión Proteica , ARN Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMEN
Coronavirus disease 2019 (COVID-19) pathogenesis is influenced by reactive oxygen species (ROS). Nevertheless, the precise mechanisms implicated remain poorly understood. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the main driver for this condition, is a structural protein indispensable for viral replication and assembly, and its role in ROS production has not been reported. This study shows that SARS-CoV-2 N protein expression enhances mitochondrial ROS level. Bulk RNA-sequencing suggests of aberrant redox state of the electron transport chain. Accordingly, this protein hinders ATP production but simultaneously augments the activity of complexes I and III, and most mitochondrially encoded complex I and III proteins are upregulated by it. Mechanistically, N protein of SARS-CoV-2 shows significant mitochondrial localization. It interacts with mitochondrial transcription components and stabilizes them. Moreover, it also impairs the activity of antioxidant enzymes with or without detectable interaction.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Especies Reactivas de Oxígeno , Proteínas de la Nucleocápside/química , Replicación ViralRESUMEN
SUMMARYNegative and ambisense RNA viruses are the causative agents of important human diseases such as influenza, measles, Lassa fever, and Ebola hemorrhagic fever. The viral genome of these RNA viruses consists of one or more single-stranded RNA molecules that are encapsidated by viral nucleocapsid proteins to form a ribonucleoprotein complex (RNP). This RNP acts as protection, as a scaffold for RNA folding, and as the context for viral replication and transcription by a viral RNA polymerase. However, the roles of the viral nucleoproteins extend beyond these functions during the viral infection cycle. Recent advances in structural biology techniques and analysis methods have provided new insights into the formation, function, dynamics, and evolution of negative sense virus nucleocapsid proteins, as well as the role that they play in host innate immune responses against viral infection. In this review, we discuss the various roles of nucleocapsid proteins, both in the context of RNPs and in RNA-free states, as well as the open questions that remain.
Asunto(s)
Virus ARN , Virosis , Humanos , Virus ARN/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , ARN Viral/química , Replicación Viral/fisiología , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismoRESUMEN
The human immunodeficiency virus-1 (HIV-1) nucleocapsid protein (NCp7) is a nucleic acid chaperone protein with two highly conserved zinc fingers. To exert its key roles in the viral cycle, NCp7 interacts with several host proteins. Among them, the human NoL12 protein (hNoL12) was previously identified in genome wide screens as a potential partner of NCp7. hNoL12 is a highly conserved 25 kDa nucleolar RNA-binding protein implicated in the 5'end processing of ribosomal RNA in the nucleolus and thus in the assembly and maturation of ribosomes. In this work, we confirmed the NCp7/hNoL12 interaction in cells by Förster resonance energy transfer visualized by Fluorescence Lifetime Imaging Microscopy and co-immunoprecipitation. The interaction between NCp7 and hNoL12 was found to strongly depend on their both binding to RNA, as shown by the loss of interaction when the cell lysates were pretreated with RNase. Deletion mutants of hNoL12 were tested for their co-immunoprecipitation with NCp7, leading to the identification of the exonuclease domain of hNoL12 as the binding domain for NCp7. Finally, the interaction with hNoL12 was found to be specific of the mature NCp7 and to require NCp7 basic residues. IMPORTANCE HIV-1 mature nucleocapsid (NCp7) results from the maturation of the Gag precursor in the viral particle and is thus mostly abundant in the first phase of the infection which ends with the genomic viral DNA integration in the cell genome. Most if not all the nucleocapsid partners identified so far are not specific of the mature form. We described here the specific interaction in the nucleolus between NCp7 and the human nucleolar protein 12, a protein implicated in ribosomal RNA maturation and DNA damage response. This interaction takes place in the cell nucleolus, a subcellular compartment where NCp7 accumulates. The absence of binding between hNoL12 and Gag makes hNoL12 one of the few known specific cellular partners of NCp7.
Asunto(s)
VIH-1 , Proteínas Nucleares , Proteínas de la Nucleocápside , Proteínas de Unión al ARN , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , Humanos , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/genética , VIH-1/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/metabolismo , Dedos de Zinc , Transferencia Resonante de Energía de Fluorescencia , Unión Proteica , InmunoprecipitaciónRESUMEN
BACKGROUND: The high prevalence and mortality rate of coronavirus disease 2019 (COVID-19) is a major global concern. Bioinformatics approaches have helped to develop new strategies to combat infectious agents, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Indeed, the structural proteins of microorganisms provide suitable epitopes for the development of vaccines to prevent infectious diseases. OBJECTIVES: The present study aimed to use bioinformatics tools to find peptides from the membrane (M) and nucleocapsid (N) proteins with effective cellular and humoral immunogenicity. MATERIAL AND METHODS: Sequences of the M and N proteins were sourced from the National Center for Biotechnology Information (NCBI). The conserved regions of the proteins with the highest immunogenicity were identified and assessed using different servers, and the physicochemical and biochemical properties of the epitopes were evaluated. Finally, allergenicity, antigenicity and docking to human leukocyte antigen (HLA) were investigated. RESULTS: The data indicated that the best epitopes were LVIGFLFLT and LFLTWICLL (as membrane epitopes), and KLDDKDPNFKDQ (as a nucleocapsid epitope), with significant immunogenicity and no evidence of allergenicity. The 3 epitopes are stable peptides that can interact with HLA to induce strong immune responses. CONCLUSIONS: The findings indicate that 3 common epitopes could effectively elicit an immune response against the disease. Hence, in vitro and in vivo studies are recommended to confirm the theoretical information.
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COVID-19 , Vacunas Virales , Humanos , SARS-CoV-2 , COVID-19/prevención & control , Proteínas de la Nucleocápside/química , Vacunas contra la COVID-19 , Vacunas Virales/química , Epítopos de Linfocito T/química , PéptidosRESUMEN
There is still much to uncover regarding the molecular details of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. As the most abundant protein, coronavirus nucleocapsid (N) protein encapsidates viral RNAs, serving as the structural component of ribonucleoprotein and virion, and participates in transcription, replication, and host regulations. Virus-host interaction might give clues to better understand how the virus affects or is affected by its host during infection and identify promising therapeutic candidates. Considering the critical roles of N, we here established a new cellular interactome of SARS-CoV-2 N by using a high-specific affinity purification (S-pulldown) assay coupled with quantitative mass spectrometry and immunoblotting validations, uncovering many N-interacting host proteins unreported previously. Bioinformatics analysis revealed that these host factors are mainly involved in translation regulations, viral transcription, RNA processes, stress responses, protein folding and modification, and inflammatory/immune signaling pathways, in line with the supposed actions of N in viral infection. Existing pharmacological cellular targets and the directing drugs were then mined, generating a drug-host protein network. Accordingly, we experimentally identified several small-molecule compounds as novel inhibitors against SARS-CoV-2 replication. Furthermore, a newly identified host factor, DDX1, was verified to interact and colocalize with N mainly by binding to the N-terminal domain of the viral protein. Importantly, loss/gain/reconstitution-of-function experiments showed that DDX1 acts as a potent anti-SARS-CoV-2 host factor, inhibiting the viral replication and protein expression. The N-targeting and anti-SARS-CoV-2 abilities of DDX1 are consistently independent of its ATPase/helicase activity. Further mechanism studies revealed that DDX1 impedes multiple activities of N, including the N-N interaction, N oligomerization, and N-viral RNA binding, thus likely inhibiting viral propagation. These data provide new clues to better depiction of the N-cell interactions and SARS-CoV-2 infection and may help inform the development of new therapeutic candidates.
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COVID-19 , SARS-CoV-2 , Animales , Humanos , Chlorocebus aethiops , SARS-CoV-2/metabolismo , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Células Vero , Replicación Viral , ARN ViralRESUMEN
Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most threatening pathogens of swine. The nucleocapsid (N) protein is the major structural protein of the virus and has been used as a PRRSV diagnostic antigen due to its high level of inherent immunogenicity. Methods: The recombinant PRRSV N protein was generated by the prokaryotic expressing system and used to immunized mice. Monoclonal antibodies against PRRSV were produced and validated by western blot analysis and indirect immunofluorescence analysis. In this study, the linear epitope of a specific monoclonal antibody mAb (N06) was subsequently identified by enzyme-linked immunosorbent assays (ELISA) using the synthesized overlapping peptides as antigens. Results: According to the results of western blot analysis and indirect immunofluorescence analysis, mAb (N06) was capable of recognizing the native form as well as the denatured form of PRRSV N protein. The results of ELISA showed that mAb N06 recognized the epitope NRKKNPEKPHFPLATE, which was consistent with BCPREDS predictions of antigenicity. Conclusion: All the data suggested that the mAb (N06) can be used as diagnostic reagents for PRRSV detection, while the recognized linear epitope can be useful in epitope-based vaccines development, which is helpful for the control of local PRRSV infections in swine.
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Epítopos de Linfocito B , Proteínas de la Nucleocápside , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Ratones , Ratones Endogámicos BALB C , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Anticuerpos Monoclonales/inmunología , Vacunas Virales/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/diagnóstico , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológicoRESUMEN
Suramin was originally used as an antiparasitic drug in clinics. Here, we demonstrate that suramin can bind to the N-terminal domain of SARS-CoV-2 nucleocapsid protein (N-NTD) and disturb its interaction with RNA. The BLI experiments showed that N-NTD interacts suramin with a dissociate constant (Kd = 2.74 µM) stronger than that of N-NTD with ssRNA-16 (Kd = 8.37 µM). Furthermore, both NMR titration experiments and molecular docking analysis suggested that suramin mainly binds to the positively charged cavity between the finger and the palm subdomains of N-NTD, and residues R88, R92, R93, I94, R95, K102 and A156 are crucial for N-NTD capturing suramin. Besides, NMR dynamics experiments showed that suramin-bound N-NTD adopts a more rigid structure, and the loop between ß2-ß3 exhibits fast motion on the ps-ns timescale, potentially facilitating suramin binding. Our findings not only reveal the molecular basis of suramin disturbing the association of SARS-CoV-2 N-NTD with RNA but also provide valuable structural information for the development of drugs against SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Suramina/farmacología , Proteínas de la Nucleocápside/química , Simulación del Acoplamiento Molecular , Modelos Moleculares , ARN Viral/genéticaRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic is transmitted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has affected millions of people all around the world, leading to more than 6.5 million deaths. The nucleocapsid (N) phosphoprotein plays important roles in modulating viral replication and transcription, virus-infected cell cycle progression, apoptosis, and regulation of host innate immunity. As an immunodominant protein, N protein induces strong humoral and cellular immune responses in COVID-19 patients, making it a key marker for studying N-specific B cell and T cell responses and the development of diagnostic serological assays and efficient vaccines. In this review, we focus on the structural and functional features and the kinetic and epitope mapping of B cell and T cell responses against SARS-CoV-2 N protein to extend our understanding on the development of sensitive and specific diagnostic immunological tests and effective vaccines.