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1.
Sci Rep ; 14(1): 5870, 2024 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-38467657

RESUMO

The nucleocapsid (N) protein of SARS-CoV-2 is known to participate in various host cellular processes, including interferon inhibition, RNA interference, apoptosis, and regulation of virus life cycles. Additionally, it has potential as a diagnostic antigen and/or immunogen. Our research focuses on examining structural changes caused by mutations in the N protein. We have modeled the complete tertiary structure of native and mutated forms of the N protein using Alphafold2. Notably, the N protein contains 3 disordered regions. The focus was on investigating the impact of mutations on the stability of the protein's dimeric structure based on binding free energy calculations (MM-PB/GB-SA) and RMSD fluctuations after MD simulations. The results demonstrated that 28 mutations out of 37 selected mutations analyzed, compared with wild-type N protein, resulted in a stable dimeric structure, while 9 mutations led to destabilization. Our results are important to understand the tertiary structure of the N protein dimer of SARS-CoV-2 and the effect of mutations on it, their behavior in the host cell, as well as for the research of other viruses belonging to the same genus additionally, to anticipate potential strategies for addressing this viral illness․.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , COVID-19/genética , Proteínas do Nucleocapsídeo/metabolismo , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Mutação
2.
Vet Microbiol ; 291: 110033, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38432077

RESUMO

The genomes of many pathogens contain high-CpG content, which is less common in most vertebrate host genomes. Such a distinct di-nucleotide composition in a non-self invader constitutes a special feature recognized by its host's immune system. The zinc-finger antiviral protein (ZAP) is part of the pattern recognition receptors (PRRs) that recognize CpG-rich viral RNA and subsequently initiate RNA degradation as an antiviral defense measure. To counteract such ZAP-mediated restriction, some viruses evolve to either suppress the CpG content in their genome or produce an antagonistic factor to evade ZAP sensing. We have previously shown that a coronavirus, Porcine epidermic diarrhea virus (PEDV), employs its nucleocapsid protein (PEDV-N) to suppress the ZAP-dependent antiviral activity. Here, we propose a mechanism by which PEDV-N suppresses ZAP function by interfering with the interaction between ZAP and its essential cofactor, Tripartite motif-containing protein 25 (TRIM25). PEDV-N was found to interact with ZAP through its N-terminal domain and with TRIM25 through its C-terminal domain. We showed that PEDV-N and ZAP compete for binding to the SPla and the RYanodine Receptor (SPRY) domain of TRIM25, resulting in PEDV-N preventing TRIM25 from interacting with and promoting ZAP. Our result also showed that the presence of PEDV-N in the complex reduces the E3 ligase activity of TRIM25 on ZAP, which is required for the antiviral activity of ZAP. The host-pathogen interaction mechanism presented herein provides an insight into the new function of this abundant and versatile viral protein from a coronavirus which could be a key target for development of antiviral interventions.


Assuntos
Ubiquitina-Proteína Ligases , Vírus , Animais , Suínos , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Antivirais/farmacologia , Antivirais/metabolismo , Nucleocapsídeo , Zinco
3.
Spectrochim Acta A Mol Biomol Spectrosc ; 311: 123977, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38310743

RESUMO

A rapid, simple, sensitive, and selective point-of-care diagnosis tool kit is vital for detecting the coronavirus disease (COVID-19) based on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain. Currently, the reverse transcriptase-polymerase chain reaction (RT-PCR) is the best technique to detect the disease. Although a good sensitivity has been observed in RT-PCR, the isolation and screening process for high sample volume is limited due to the time-consuming and laborious work. This study introduced a nucleic acid-based surface-enhanced Raman scattering (SERS) sensor to detect the nucleocapsid gene (N-gene) of SARS-CoV-2. The Raman scattering signal was amplified using gold nanoparticles (AuNPs) possessing a rod-like morphology to improve the SERS effect, which was approximately 12-15 nm in diameter and 40-50 nm in length. These nanoparticles were functionalised with the single-stranded deoxyribonucleic acid (ssDNA) complemented with the N-gene. Furthermore, the study demonstrates method selectivity by strategically testing the same virus genome at different locations. This focused approach showcases the method's capability to discern specific genetic variations, ensuring accuracy in viral detection. A multivariate statistical analysis technique was then applied to analyse the raw SERS spectra data using the principal component analysis (PCA). An acceptable variance amount was demonstrated by the overall variance (82.4 %) for PC1 and PC2, which exceeded the desired value of 80 %. These results successfully revealed the hidden information in the raw SERS spectra data. The outcome suggested a more significant thymine base detection than other nitrogenous bases at wavenumbers 613, 779, 1219, 1345, and 1382 cm-1. Adenine was also less observed at 734 cm-1, and ssDNA-RNA hybridisations were presented in the ketone with amino base SERS bands in 1746, 1815, 1871, and 1971 cm-1 of the fingerprint. Overall, the N-gene could be detected as low as 0.1 nM within 10 mins of incubation time. This approach could be developed as an alternative point-of-care diagnosis tool kit to detect and monitor the COVID-19 disease.


Assuntos
COVID-19 , Nanopartículas Metálicas , Nanotubos , Ácidos Nucleicos , Humanos , Análise Espectral Raman/métodos , Ouro , Análise de Componente Principal , SARS-CoV-2/genética , COVID-19/diagnóstico , Nucleocapsídeo
4.
Anal Chim Acta ; 1292: 342241, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38309851

RESUMO

In addition to confirming virus infection, quantitative identification of the antibodies to severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) also evaluates persons immunity to guide personal protection. However, portable assays for fast and accurate quantification of SARS-CoV-2 antibodies remain challenging. In this work, we synthesized Au@Pt star-like nanoparticles (NPs) quickly and easily by a one-pot wet-chemical approach, allowing the stellate Au core to be partially decorated by Pt nanoshells. The nanoparticles were used as probe in a lateral flow immunoassay (LFIA) that operated in both colorimetric and photothermal dual modes, which could detect the antibodies to the SARS-CoV-2 nucleocapsid (N) protein with high sensitivity. Due to the sharp tips on the external region of nanostars and surface plasmon coupling effect between the Au core and Pt shell, the NIR absorption capacity and photothermal performance of these NPs were exceptional. Under optimal conditions, the colorimetric mode's detection limit for SARS-CoV-2 N protein antibody was 1 ng mL-1, which is significantly lower by 2-order of magnitude compared to commercially available colloidal gold strips. And the detection limit for the photothermal mode was as low as 24.91 pg mL-1, which was approximately 40-fold more sensitive than colorimetric detection. Moreover, the method demonstrated favorable specificity, reproducibility and stability. Finally, the approach was employed for the successful identification of actual serum samples. Therefore, the dual-mode LFIA can be applied for screening and tracking the early immunological reaction to SARS-CoV-2, and it has great promise for clinical application.


Assuntos
COVID-19 , Nanopartículas Metálicas , Nanoconchas , Humanos , SARS-CoV-2 , Colorimetria , Reprodutibilidade dos Testes , COVID-19/diagnóstico , Anticorpos Antivirais , Imunoensaio , Nucleocapsídeo
5.
Front Immunol ; 15: 1332440, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38375473

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus to cause acute respiratory distress syndrome (ARDS) and contains four structural proteins: spike, envelope, membrane, and nucleocapsid. An increasing number of studies have demonstrated that all four structural proteins of SARS-CoV-2 are capable of causing lung injury, even without the presence of intact virus. Therefore, the topic of SARS-CoV-2 structural protein-evoked lung injury warrants more attention. In the current article, we first synopsize the structural features of SARS-CoV-2 structural proteins. Second, we discuss the mechanisms for structural protein-induced inflammatory responses in vitro. Finally, we list the findings that indicate structural proteins themselves are toxic and sufficient to induce lung injury in vivo. Recognizing mechanisms of lung injury triggered by SARS-CoV-2 structural proteins may facilitate the development of targeted modalities in treating COVID-19.


Assuntos
COVID-19 , Lesão Pulmonar , Humanos , SARS-CoV-2/metabolismo , Nucleocapsídeo/metabolismo , Proteínas do Envelope Viral/metabolismo
6.
J Med Virol ; 96(1): e29358, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38180230

RESUMO

In hospitalized children, SARS-CoV-2 infection can present as either a primary reason for admission (patients admitted for COVID-19) or an incidental finding during follow-up (patients admitted with COVID-19). We conducted a nested case-control study within a cohort of pediatric patients with confirmed SARS-CoV-2 infection, to investigate the concentration of plasma nucleocapsid antigen (N-Ag) in children admitted for COVID-19 or with COVID-19. While reverse transcriptase polymerase chain reaction Ct values in nasopharyngeal swab were similar between the two groups, children admitted for COVID-19 had a higher rate of detectable N-Ag (12/18 (60.7%) versus 6/18 (33.3%), p = 0.0455) and a higher concentration of N-Ag (medians: 19.51 g/mL vs. 1.08 pg/mL, p = 0.0105). In children hospitalized for COVID-19, the youngest had higher concentration of N-Ag (r = -0.74, p = 0.0004). We also observed a lower prevalence of detectable spike antibodies in children hospitalized for COVID-19 compared to those hospitalized for other medical reasons (3/15 [20%] vs. 13/16 [81.25%], respectively, p = < 0.0011), but similar rates of IgG nucleocapsid antibodies (5/14 [35.7%] vs. 6/17 [35.3%], respectively, p = 0.99). Our findings indicate that N-Ag is associated with COVID-19-related hospitalizations in pediatric patients, and less frequently detected in children tested positive for SARS-CoV-2 but hospitalized for another medical reason. Further studies are needed to confirm the value of N-Ag in identifying COVID-19 disease infections in which SARS-CoV-2 is the main pathogen responsible for symptoms.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Criança , Estudos de Casos e Controles , COVID-19/diagnóstico , Nucleocapsídeo , Vírion , Antígenos Virais , Imunoglobulina G
7.
Sci Adv ; 10(2): eadi7606, 2024 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-38198557

RESUMO

Nuclear import of the hepatitis B virus (HBV) nucleocapsid is essential for replication that occurs in the nucleus. The ~360-angstrom HBV capsid translocates to the nuclear pore complex (NPC) as an intact particle, hijacking human importins in a reaction stimulated by host kinases. This paper describes the mechanisms of HBV capsid recognition by importins. We found that importin α1 binds a nuclear localization signal (NLS) at the far end of the HBV coat protein Cp183 carboxyl-terminal domain (CTD). This NLS is exposed to the capsid surface through a pore at the icosahedral quasi-sixfold vertex. Phosphorylation at serine-155, serine-162, and serine-170 promotes CTD compaction but does not affect the affinity for importin α1. The binding of 30 importin α1/ß1 augments HBV capsid diameter to ~620 angstroms, close to the maximum size trafficable through the NPC. We propose that phosphorylation favors CTD externalization and prompts its compaction at the capsid surface, exposing the NLS to importins.


Assuntos
Vírus da Hepatite B , Nucleocapsídeo , Humanos , Transporte Ativo do Núcleo Celular , Carioferinas , Proteínas do Capsídeo , Fatores Imunológicos , Serina
8.
Anal Chim Acta ; 1287: 342070, 2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38182376

RESUMO

BACKGROUND: Early diagnosis of SARS-CoV-2 infection is still critical to control COVID-19 outbreak. Traditional polymerase chain reaction, enzyme-linked immunosorbent assay or lateral flow immunoassay performed poorly on detection times, sample preparation process and accuracy. Surface-enhanced Raman scattering (SERS)-based detection has emerged as a powerful analytical technique, which overcomes the above limitations. However, due to the near-field effect of traditional substrate, it is difficult to monitor the binding event of aptamers with proteins. It is obvious that a novel SERS substrate thatsupportedextended and stronger electromagnetic fields was required to hold long-range effects and allow for binding event testing. RESULTS: Driven by this challenge, we reported a long-range SERS-active substrate, which was built by inserting bowtie nanoaperture arrays in a refractive-index-symmetric environment and Au mirror surfaces, for SARS-CoV-2 protein binding event detection. Then, a double-π structure aptasensor was simply designed through the hybridization of spike (S) and nucleocapsid (N) proteins aptamers, and a corresponding complementary strand. This kind of double-π structure would dissociate when targets proteins S and N existed and led to the SERS responses decreased, which established the detection basis of our system. What's more, due to two Raman labels were involved, both proteins S and N can be sensed simultaneously. Our proposed method showed improved sensitivity with a low limit of detection for multiplex detection (1.6 × 10-16 g/mL for protein S and 1.0 × 10-16 g/mL for protein N) over a wide concentration range. SIGNIFICANCE: This represents the first long-range SERS apatasensor platform for detection of S and N proteins simultaneously. Our method showed high sensitivity, selectivity, reproducibility, stability and remarkable recoveries in human in saliva and serum samples, which is particularly important for the early diagnostics of COVID as well as for future unknown coronavirus.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Reprodutibilidade dos Testes , COVID-19/diagnóstico , Nucleocapsídeo , Campos Eletromagnéticos , Oligonucleotídeos
9.
Nat Commun ; 15(1): 250, 2024 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-38177118

RESUMO

Baculoviruses are insect-infecting pathogens with wide applications as biological pesticides, in vitro protein production vehicles and gene therapy tools. Its cylindrical nucleocapsid, which encapsulates and protects the circular double-stranded viral DNA encoding proteins for viral replication and entry, is formed by the highly conserved major capsid protein VP39. The mechanism for VP39 assembly remains unknown. We use electron cryomicroscopy to determine a 3.2 Å helical reconstruction of an infectious nucleocapsid of Autographa californica multiple nucleopolyhedrovirus, revealing how dimers of VP39 assemble into a 14-stranded helical tube. We show that VP39 comprises a distinct protein fold conserved across baculoviruses, which includes a Zinc finger domain and a stabilizing intra-dimer sling. Analysis of sample polymorphism shows that VP39 assembles in several closely-related helical geometries. This VP39 reconstruction reveals general principles for baculoviral nucleocapsid assembly.


Assuntos
Baculoviridae , Nucleocapsídeo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Spodoptera , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo
10.
Clin Exp Immunol ; 215(3): 268-278, 2024 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-37313783

RESUMO

As there are limited data on B-cell epitopes for the nucleocapsid protein in SARS-CoV-2, we sought to identify the immunodominant regions within the N protein, recognized by patients with varying severity of natural infection with the Wuhan strain (WT), delta, omicron, and in those who received the Sinopharm vaccines, which is an inactivated, whole virus vaccine. Using overlapping peptides representing the N protein, with an in-house ELISA, we mapped the immunodominant regions within the N protein, in seronegative (n = 30), WT infected (n = 30), delta infected (n = 30), omicron infected + vaccinated (n = 20) and Sinopharm (BBIBP-CorV) vaccinees (n = 30). We then investigated the sensitivity and specificity of these immunodominant regions and analyzed their conservation with other SARS-CoV-2 variants of concern, seasonal human coronaviruses, and bat Sarbecoviruses. We identified four immunodominant regions aa 29-52, aa 155-178, aa 274-297, and aa 365-388, which were highly conserved within SARS-CoV-2 and the bat coronaviruses. The magnitude of responses to these regions varied based on the infecting SARS-CoV-2 variants, >80% of individuals gave responses above the positive cut-off threshold to many of the four regions, with some differences with individuals who were infected with different VoCs. These regions were found to be 100% specific, as none of the seronegative individuals gave any responses. As these regions were highly specific with high sensitivity, they have a potential to be used to develop diagnostic assays and to be used in development of vaccines.


Assuntos
COVID-19 , Quirópteros , Humanos , Animais , SARS-CoV-2 , Formação de Anticorpos , Epitopos Imunodominantes , Nucleocapsídeo , Anticorpos Antivirais
11.
Vaccine ; 42(5): 1051-1064, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-37816655

RESUMO

SARS-CoV-2, severe acute respiratory syndrome coronavirus-2, causes coronavirus disease- 2019 (COVID-19). Mostly, COVID-19 causes respiratory symptoms that can resemble those of a cold, the flu, or pneumonia. COVID-19 may harm more than just lungs and respiratory systems. It may also have an impact on other parts of the body and debilitating effects on humans, necessitating the development of vaccines at an unprecedented rate in order to protect humans from infections. In response to SARS-CoV-2 infection, mRNA, viral vector-based carrier and inactivated virus-based vaccines, as well as subunit vaccines, have recently been developed. We developed Relcovax®, a dual antigen (Receptor binding domain (RBD) and Nucleocapsid (N) proteins) subunit protein vaccine candidate. Preliminary mouse preclinical studies revealed that Relcovax® stimulates cell-mediated immunity and provides broader protection against two SARS-CoV-2 variants, including the delta strain. Before conducting human studies, detailed preclinical safety assessments are required, so Relcovax® was tested for safety, and immunogenicity in 28-day repeated dose toxicity studies in rats and rabbits. In the toxicity studies, there were no mortality or morbidity, abnormal clinical signs, abnormalities in a battery of neurobehavioral observations, abnormalities in detailed clinical and ophthalmological examinations, or changes in body weights or feed consumption. In any of the studies, no abnormal changes in organ weights, haematology, clinical chemistry, urinalysis parameters, or pathological findings were observed. Immunogenicity tests on rats and rabbits revealed 100 % seroconversion. Relcovax® was therefore found to be safe in animals, with a No Observed Adverse Effect Level (NOAEL) of 20 µg/protein in rats and rabbits. In efficacy studies, Relcovax® immunised hamsters demonstrated dose-dependent protection against SARS-CoV-2 infection, with a high dose (20 µg/protein) being the most protective, while in cynomolgus macaque monkey study, lowest dose 5 µg/protein had the highest efficacy. In conclusion, Relcovax® was found to be safe, immunogenic, and efficacious in in vivo studies.


Assuntos
COVID-19 , Vacinas de Subunidades , Animais , Cricetinae , Humanos , Camundongos , Coelhos , Ratos , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Imunogenicidade da Vacina , Nucleocapsídeo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vacinas de Subunidades/efeitos adversos , Vacinas Virais
12.
PLoS Pathog ; 19(12): e1011832, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38039340

RESUMO

After entry into cells, herpes simplex virus (HSV) nucleocapsids dock at nuclear pore complexes (NPCs) through which viral genomes are released into the nucleoplasm where viral gene expression, genome replication, and early steps in virion assembly take place. After their assembly, nucleocapsids are translocated to the cytoplasm for final virion maturation. Nascent cytoplasmic nucleocapsids are prevented from binding to NPCs and delivering their genomes to the nucleus from which they emerged, but how this is accomplished is not understood. Here we report that HSV pUL16 and pUL21 deletion mutants accumulate empty capsids at the cytoplasmic face of NPCs late in infection. Additionally, prior expression of pUL16 and pUL21 prevented incoming nucleocapsids from docking at NPCs, delivering their genomes to the nucleus and initiating viral gene expression. Both pUL16 and pUL21 localized to the nuclear envelope, placing them in an appropriate location to interfere with nucleocapsid/NPC interactions.


Assuntos
Herpes Simples , Herpesvirus Humano 1 , Humanos , Capsídeo/metabolismo , Poro Nuclear/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Nucleocapsídeo/metabolismo
13.
Sci Adv ; 9(50): eadj9974, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-38100595

RESUMO

Influenza virus genome encapsidation is essential for the formation of a helical viral ribonucleoprotein (vRNP) complex composed of nucleoproteins (NP), the trimeric polymerase, and the viral genome. Although low-resolution vRNP structures are available, it remains unclear how the viral RNA is encapsidated and how NPs assemble into the helical filament specific of influenza vRNPs. In this study, we established a biological tool, the RNP-like particles assembled from recombinant influenza A virus NP and synthetic RNA, and we present the first subnanometric cryo-electron microscopy structure of the helical NP-RNA complex (8.7 to 5.3 Å). The helical RNP-like structure reveals a parallel double-stranded conformation, allowing the visualization of NP-NP and NP-RNA interactions. The RNA, located at the interface of neighboring NP protomers, interacts with conserved residues previously described as essential for the NP-RNA interaction. The NP undergoes conformational changes to enable RNA binding and helix formation. Together, our findings provide relevant insights for understanding the mechanism for influenza genome encapsidation.


Assuntos
Influenza Humana , Nucleoproteínas , Humanos , Nucleoproteínas/química , Microscopia Crioeletrônica , Ribonucleoproteínas/genética , RNA Viral/metabolismo , Nucleocapsídeo/metabolismo
14.
Viruses ; 15(12)2023 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-38140645

RESUMO

From the first isolation of the cystovirus bacteriophage Φ6 from Pseudomonas syringae 50 years ago, we have progressed to a better understanding of the structure and transformations of many parts of the virion. The three-layered virion, encapsulating the tripartite double-stranded RNA (dsRNA) genome, breaches the cell envelope upon infection, generates its own transcripts, and coopts the bacterial machinery to produce its proteins. The generation of a new virion starts with a procapsid with a contracted shape, followed by the packaging of single-stranded RNA segments with concurrent expansion of the capsid, and finally replication to reconstitute the dsRNA genome. The outer two layers are then added, and the fully formed virion released by cell lysis. Most of the procapsid structure, composed of the proteins P1, P2, P4, and P7 is now known, as well as its transformations to the mature, packaged nucleocapsid. The outer two layers are less well-studied. One additional study investigated the binding of the host protein YajQ to the infecting nucleocapsid, where it enhances the transcription of the large RNA segment that codes for the capsid proteins. Finally, I relate the structural aspects of bacteriophage Φ6 to those of other dsRNA viruses, noting the similarities and differences.


Assuntos
Bacteriófago phi 6 , Bacteriófagos , Animais , RNA Viral/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , Nucleocapsídeo/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , RNA de Cadeia Dupla/metabolismo , Estágios do Ciclo de Vida
15.
Viruses ; 15(12)2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-38140607

RESUMO

Hepatitis B virus (HBV) is the primary contributor to severe liver ailments, encompassing conditions such as cirrhosis and hepatocellular carcinoma. Globally, 257 million people are affected by HBV annually and 887,000 deaths are attributed to it, representing a substantial health burden. Regrettably, none of the existing therapies for chronic hepatitis B (CHB) have achieved satisfactory clinical cure rates. This issue stems from the existence of covalently closed circular DNA (cccDNA), which is difficult to eliminate from the nucleus of infected hepatocytes. HBV genetic material is composed of partially double-stranded DNA that forms complexes with viral polymerase inside an icosahedral capsid composed of a dimeric core protein. The HBV core protein, consisting of 183 to 185 amino acids, plays integral roles in multiple essential functions within the HBV replication process. In this review, we describe the effects of sulfamoyl-based carboxamide capsid assembly modulators (CAMs) on capsid assembly, which can suppress HBV replication and disrupt the production of new cccDNA. We present research on classical, first-generation sulfamoyl benzocarboxamide CAMs, elucidating their structural composition and antiviral efficacy. Additionally, we explore newly identified sulfamoyl-based CAMs, including sulfamoyl bicyclic carboxamides, sulfamoyl aromatic heterocyclic carboxamides, sulfamoyl aliphatic heterocyclic carboxamides, cyclic sulfonamides, and non-carboxamide sulfomoyl-based CAMs. We believe that certain molecules derived from sulfamoyl groups have the potential to be developed into essential components of a well-suited combination therapy, ultimately yielding superior clinical efficacy outcomes in the future.


Assuntos
Hepatite B Crônica , Hepatite B , Humanos , Vírus da Hepatite B/metabolismo , Antivirais/uso terapêutico , Nucleocapsídeo/metabolismo , Hepatite B Crônica/tratamento farmacológico , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , DNA Circular/genética , DNA Circular/metabolismo , Replicação Viral , DNA Viral/genética , DNA Viral/metabolismo , Hepatite B/tratamento farmacológico , Hepatite B/metabolismo
16.
J Med Virol ; 95(11): e29229, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37966995

RESUMO

Antibody assays with the nucleocapsid (NC) protein as the target antigen can identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections when polymerase chain reaction (PCR) analyses are unavailable. Regarding the kinetics of NC-specific antibodies, vaccine breakthroughs with Omicron subvariants may differ from infections with the ancestral wild-type virus. Therefore, we evaluated which assays have the highest sensitivity for detecting NC-specific antibodies after various intervals since breakthrough infections with an Omicron subvariant. The study included 279 samples from vaccinated subjects who experienced PCR-confirmed Omicron breakthrough infections between 21 and 266 days before sampling. The samples were comparatively assessed with the Elecsys® Anti-SARS-CoV-2 N (Roche), the Anti-SARS-CoV-2-NCP-ELISA (Euroimmun), the recomLine SARS-CoV-2 IgG (Mikrogen), and the SARS-CoV-2 ViraChip IgG assays (Viramed). In the whole cohort, the Elecsys® Anti-SARS-CoV-2 N assay displayed the highest sensitivity (93%, p < 0.0001), followed by the recomLine SARS-CoV-2 IgG assay (70%), the SARS-CoV-2 ViraChip IgG assay (41%) and the Anti-SARS-CoV-2-NCP-ELISA (35%). Although measured antibody levels and time-dependent sensitivities differed, the extent of the antibody decrease was similar among all assays. As demonstrated by this study, manufacturer-dependent differences in the sensitivities of NC-specific antibody assays should be considered when serology is applied to link previous SARS-CoV-2 infections with potential post-COVID sequelae.


Assuntos
Vacinas contra COVID-19 , COVID-19 , Humanos , SARS-CoV-2 , COVID-19/diagnóstico , COVID-19/prevenção & controle , Nucleocapsídeo , Anticorpos Antivirais , Imunoglobulina G , Proteínas do Nucleocapsídeo , Infecções Irruptivas , Sensibilidade e Especificidade
17.
Nat Commun ; 14(1): 7481, 2023 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-37980340

RESUMO

Baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used as a bioinsecticide and a protein expression vector. Despite their importance, very little is known about the structure of most baculovirus proteins. Here, we show a 3.2 Å resolution structure of helical cylindrical body of the AcMNPV nucleocapsid, composed of VP39, as well as 4.3 Å resolution structures of both the head and the base of the nucleocapsid composed of over 100 protein subunits. AcMNPV VP39 demonstrates some features of the HK97-like fold and utilizes disulfide-bonds and a set of interactions at its C-termini to mediate nucleocapsid assembly and stability. At both ends of the nucleocapsid, the VP39 cylinder is constricted by an outer shell ring composed of proteins AC104, AC142 and AC109. AC101(BV/ODV-C42) and AC144(ODV-EC27) form a C14 symmetric inner layer at both capsid head and base. In the base, these proteins interact with a 7-fold symmetric capsid plug, while a portal-like structure is seen in the central portion of head. Additionally, we propose an application of AlphaFold2 for model building in intermediate resolution density.


Assuntos
Baculoviridae , Nucleocapsídeo , Animais , Baculoviridae/metabolismo , Microscopia Crioeletrônica , Spodoptera , Nucleocapsídeo/metabolismo , Proteínas do Capsídeo/metabolismo
18.
Arch Biochem Biophys ; 750: 109820, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37956938

RESUMO

The nucleocapsid (N) protein of SARS-CoV-2 plays a pivotal role in encapsulating the viral genome. Developing antiviral treatments for SARS-CoV-2 is imperative due to the diminishing immunity of the available vaccines. This study targets the RNA-binding site located in the N-terminal domain (NTD) of the N-protein to identify the potential antiviral molecules against SARS-CoV-2. A structure-based repurposing approach identified the twelve high-affinity molecules from FDA-approved drugs, natural products, and the LOPAC1280 compound libraries that precisely bind to the RNA binding site within the NTD. The interaction of these potential antiviral agents with the purified NTD protein was thermodynamically characterized using isothermal titration calorimetry (ITC). A fluorescence-based plate assay to assess the RNA binding inhibitory activity of small molecules against the NTD has been employed, and the selected compounds exhibited significant RNA binding inhibition with calculated IC50 values ranging from 8.8 µM to 15.7 µM. Furthermore, the antiviral efficacy of these compounds was evaluated using in vitro cell-based assays targeting the replication of SARS-CoV-2. Remarkably, two compounds, Telmisartan and BMS-189453, displayed potential antiviral activity against SARS-CoV-2, with EC50 values of approximately 1.02 µM and 0.98 µM, and a notable selective index of >98 and > 102, respectively. This study gives valuable insight into developing therapeutic interventions against SARS-CoV-2 by targeting the N-protein, a significant effort given the global public health concern posed due to the virus re-emergence and long COVID-19 disease.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Antivirais/farmacologia , Antivirais/química , Síndrome Pós-COVID-19 Aguda , Nucleocapsídeo/metabolismo , Termodinâmica , RNA , Simulação de Acoplamento Molecular
19.
Braz J Microbiol ; 54(4): 2893-2901, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37930615

RESUMO

The gold standard for diagnosing COVID-19 in the acute phase is RT-qPCR. However, this molecular technique can yield false-negative results when nasopharyngeal swab collection is not conducted during viremia. To mitigate this challenge, the enzyme-linked immunosorbent assay (ELISA) identifies anti-SARS-CoV-2 IgM antibodies in the initial weeks after symptom onset, facilitating early COVID-19 diagnosis. This study introduces a novel and highly specific IgM antibody capture ELISA (MAC-ELISA), which utilizes biotinylated recombinant SARS-CoV-2 nucleocapsid (N) antigen produced in plants. Our biotinylated approach streamlines the procedure by eliminating the requirement for an anti-N-conjugated antibody, circumventing the need for peroxidase-labeled antigens, and preventing cross-reactivity with IgM autoantibodies such as rheumatoid factor. Performance evaluation of the assay involved assessing sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy using 682 RT-qPCR-positive samples, categorized by weeks relative to symptoms onset. Negative controls included 205 pre-pandemic serum samples and 46 serum samples from patients diagnosed with other diseases. Based on a cut-off of 0.087 and ROC curve analysis, the highest sensitivity of 81.2% was observed in the 8-14 days post-symptom (dps) group (2nd week), followed by sensitivities of 73.8% and 68.37% for the 1-7 dps (1st week) and 15-21 dps groups (3rd week), respectively. Specificity was consistently 100% across all groups. This newly developed biotinylated N-MAC-ELISA offers a more streamlined and cost-effective alternative to molecular diagnostics. It enables simultaneous testing of multiple samples and effectively identifies individuals with false-negative results.


Assuntos
COVID-19 , Humanos , COVID-19/diagnóstico , Teste para COVID-19 , SARS-CoV-2 , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina M , Anticorpos Antivirais , Nucleocapsídeo , Sensibilidade e Especificidade
20.
Proc Natl Acad Sci U S A ; 120(46): e2306129120, 2023 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-37939083

RESUMO

Controlling the biodistribution of protein- and nanoparticle-based therapeutic formulations remains challenging. In vivo library selection is an effective method for identifying constructs that exhibit desired distribution behavior; library variants can be selected based on their ability to localize to the tissue or compartment of interest despite complex physiological challenges. Here, we describe further development of an in vivo library selection platform based on self-assembling protein nanoparticles encapsulating their own mRNA genomes (synthetic nucleocapsids or synNCs). We tested two distinct libraries: a low-diversity library composed of synNC surface mutations (45 variants) and a high-diversity library composed of synNCs displaying miniproteins with binder-like properties (6.2 million variants). While we did not identify any variants from the low-diversity surface library that yielded therapeutically relevant changes in biodistribution, the high-diversity miniprotein display library yielded variants that shifted accumulation toward lungs or muscles in just two rounds of in vivo selection. Our approach should contribute to achieving specific tissue homing patterns and identifying targeting ligands for diseases of interest.


Assuntos
Biblioteca de Peptídeos , Proteínas , Distribuição Tecidual , Nucleocapsídeo , Mutação
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