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Targeting a Dark Excited State of HIV-1 Nucleocapsid by Antiretroviral Thioesters Revealed by NMR Spectroscopy.

HIV-1 nucleocapsid (NCp7) is a two Cys2 HisCys zinc knuckle (N-Zn and C-Zn) protein that plays a key role in viral replication. NCp7 conformational dynamics is characterized by NMR relaxation dispersion and chemical exchange saturation transfer measurements. While the N-Zn knuckle is conformationally stable, the C-Zn knuckle interconverts on the millisecond timescale between the major state, in which the zinc is coordinated by three cysteines and a histidine, and two folded minor species (with populations around 1 %) in which one of the coordination bonds (Cys413-Sγ-Zn or His421-Nϵ2-Zn) is hyd…

Computational prediction and biochemical characterization of novel RNA aptamers to Rift Valley fever virus nucleocapsid protein.

Rift Valley fever virus (RVFV) is a potent human and livestock pathogen endemic to sub-Saharan Africa and the Arabian Peninsula that has potential to spread to other parts of the world. Although there is no proven effective and safe treatment for RVFV infections, a potential therapeutic target is the virally encoded nucleocapsid protein (N). During the course of infection, N binds to viral RNA, and perturbation of this interaction can inhibit viral replication. To gain insight into how N recognizes viral RNA specifically, we designed an algorithm that uses a distance matrix and multidimensiona…

Use of recombinant nucleocapsid proteins for serological diagnosis of feline coronavirus infection by three immunochromatographic tests.

Three types of immunochromatographic assays (ICAs) were designed to detect anti-feline coronavirus (FCoV) antibodies. Recombinant FCoV nucleocapsid protein (rNP) was used as a conjugate or test line in all 3 ICA kits (CJIgG/TNP, CJNP/TNP, and CJNP/TPA). All three ICA kits were capable of detecting anti-FCoV antibodies; however, non-specific positive reactions of anti-FCoV antibody-negative plasma samples with the test line were observed in 2 ICA kits (CJIgG/TNP and CJNP/TNP), in which rNP was used as the test line. On the other hand, the specific detection of anti-FCoV antibodies was possible …

Anti-nucleocapsid protein immune responses counteract pathogenic effects of Rift Valley fever virus infection in mice.

The known virulence factor of Rift Valley fever virus (RVFV), the NSs protein, counteracts the antiviral effects of the type I interferon response. In this study we evaluated the expression of several genes in the liver and spleen involved in innate and adaptive immunity of mice immunized with a RVFV recombinant nucleocapsid protein (recNP) combined with Alhydrogel adjuvant and control animals after challenge with wild type RVFV. Mice immunized with recNP elicited an earlier IFNβ response after challenge compared to non-immunized controls. In the acute phase of liver infection in non-immunized…

[Detection of hepatitis C markers--nucleocapsid protein, RNA and virus-specific antibodies in blood donor plasma].

Comparative study of hepatitis C virus (HCV) markers (core protein, RNA, and virus-specific antibodies) was carried out in plasma samples from 80 donors. A method based on sandwich ELISA with monoclonal antibodies to recombinant protein was developed for measuring core protein. Nucleocapsid protein was detected after various treatments of precipitates obtained after concentration of virus-containing material from plasma samples. These treatments allowed differentiation of core protein in virions, free nucleocapsids, and immune complexes circulating in peripheral blood. The minimal detectable c…

Coronavirus induced encephalomyelitis: an immunodominant CD4(+)-T cell site on the nucleocapsid protein contributes to protection.

In this communication we present clear evidence, that the N-protein of MHV-JHM contains immunodominant CD4+ T-cell sites. These sites were recognized by the immune system of virus infected Lewis rats. In previous investigations we have shown, that CD4+ T-cell lines with specificity for defined viral proteins can be selected from diseased Lewis rats and mediate protection, if transferred to otherwise lethally infected animals. To define regions of the N-protein, which are immunodominant for the T-cell response, we employed bacterially expressed N-protein and truncated subfragments of N as an an

Expression of the SARS-CoV-2 nucleocapsid (N) gene in Chlorella vulgaris via electroporation-mediated transformation.

The microalga Chlorella vulgaris is a unicellular photosynthetic microorganism with the potential of serving as a host organism for foreign genes. Utilising C. vulgaris as a host organism to express the SARS-CoV-2 structural protein, such as the nucleocapsid (N) gene, is a sustainable approach with high scalability. Here, we genetically engineered C. vulgaris with the N gene of SARS-CoV-2. Initially, a recombinant plasmid, pCAMBIA1303 harbouring the SARS-CoV-2 N gene, was transformed into C. vulgaris via electroporation. Subsequently, the transgenic C. vulgaris was cultivated in BG11 medium co…

Nanoplasmonic optical fiber sensing of SARS-CoV-2 nucleocapsid protein using an aptamer-DNA tetrahedron interface.

Optical fiber sensing carries a number of potential advantages for diagnostics and biomarker detection and monitoring, yet particular challenges persist in linking molecular recognition events to a change in the refractive index. DNA aptamers carry particular advantages as functional surface molecules on optical fibers to tailor detection of specific biomolecules, yet challenges persist around sensitivity and specificity. Diagnosis of COVID-19 through detection of nucleocapsid protein (N protein) of SARS-CoV-2 provides a classic diagnostic challenge where optical fiber-based sensing could comp…

Increased anti-nucleocapsid secretory IgA and consumption of complement component 3 in post-COVID syndrome patients.

INTRODUCTION: Post-COVID syndrome represents a major global health challenge which is characterized by immune dysregulation, although many aspects of the immune response remain incompletely understood, particularly the antibody response and the role of the complement system. We previously studied a post-COVID syndrome cohort in comparison to a COVID-recovered control cohort from Comunidad de Madrid (Spain) and found that post-COVID syndrome patients exhibited readily detectable serum anti-Nucleocapsid antibodies while showing deficient antibody production against the full-length Spike, despite…

A feline coronavirus nucleocapsid protein disrupts ZC3HAV1-viral RNA association to counteract host RNA-level restriction.

BACKGROUND: Coronaviruses have evolved intricate mechanisms to evade host RNA surveillance systems and sustain efficient replication. The feline infectious peritonitis virus (FIPV), a highly lethal feline coronavirus that causes systemic vasculitis and fatal peritonitis, represents a major challenge in veterinary medicine and serves as a valuable model for studying conserved coronavirus-host interactions. Among coronavirus structural proteins, the nucleocapsid (N) protein not only packages the viral genome but also modulates host antiviral responses, yet its role in counteracting RNA restricti…

Non-neutralizing antibodies against SARS-CoV-2 nucleocapsid protein mediate variant transcendent antibody-dependent cellular cytotoxicity.

Vaccination strategies and correlates of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have predominantly focused on the spike (S) protein and neutralizing antibodies. However, the rapid emergence of SARS-CoV-2 variants has reduced the effectiveness of spike-based vaccines and monoclonal antibodies. It remains unclear how non-neutralizing antibodies that target the nucleocapsid (N) protein contribute to protection against SARS-CoV-2 variants, especially their ability to trigger antibody effector functions. These antibodies may function by binding to infected c…

Elucidating the mechanism by which HIV-1 nucleocapsid mutations confer resistance to integrase strand transfer inhibitors.

Persons with HIV (PWH) receiving integrase (IN) strand transfer inhibitors (INSTIs) have been reported to experience virologic failure (VF) in the absence of resistance mutations in IN. We previously reported that mutations in the viral nucleocapsid (NC) are selected in the presence of the INSTI dolutegravir (DTG). Here, we show that these NC mutations accelerate the kinetics of viral DNA integration, suggesting that they limit the window of time available for INSTIs to block viral DNA integration. We find that in primary peripheral blood mononuclear cells, HIV-1 acquires mutations in the vira…

A non-spike nucleocapsid R204P mutation in SARS-CoV-2 Omicron XEC enhances inflammation and pathogenicity.

The global circulation of SARS-CoV-2 in human populations has driven the emergence of Omicron subvariants, which have become highly diversif … nicity of XEC in male hamsters is significantly higher than that of JN.1. Notably, we find that the nucleocapsid R204P mutation of XEC enhances inflammation through NF-κB activation. Recent studies suggest that the evolutionary potential of spike protein is reaching its limit. Indeed, our findings highlight the critical role of non-spike mutations in the futur …

Plasma SARS-CoV-2 nucleocapsid antigen levels are associated with lung infection and tissue-damage biomarkers.

BACKGROUND: SARS-CoV-2 nucleocapsid (N) antigen has been confirmed in the peripheral blood of patients with new coronavirus infection,yet its diagnostic and prognostic significance remains unclear. This study aimed to characterize the dynamics of SARS-CoV-2 N antigenemia in patients with novel coronavirus positivity, and to assess its potential association with clinical severity and plasma biomarker levels. METHODS: We analyzed the level of SARS-CoV-2 N antigen, spike receptor-binding domain (S-RBD) IgG, neutralizing antibodies (NAb) and tissue-damage biomarkers was assessed in 180 plasma sam…

Comparative immunobiology and cross-species validations of pan-MHC-II epitopes on Hantaan virus nucleocapsid protein.

During viral infection, CD4+ helper T-cell is indispensable for the establishment of the humoral immune protection, CTL activation, and even long-term memory response. It requires MHC-II molecules in viral structural antigens process and presentation. HTNV NP epitopes exhibited high affinity to both of HLA-II superfamilies and H-2-I genes in the present study. Immunogenicity and conservation analyses identified 34 selective epitopes, later validated by molecular docking (MD) with MHC-II structures. NP 15-mer peptides and MHC-II haplotypes were found to interact bidirectionally through hierarch

Efficient overexpression and purification of severe acute respiratory syndrome coronavirus 2 nucleocapsid proteins in Escherichia coli.

The fundamental biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducibl…

Suppression of SARS-CoV-2 nucleocapsid protein dimerization by ISGylation and its counteraction by viral PLpro.

Protein modification by the ubiquitin-like protein ISG15 (ISGylation) plays a crucial role in the immunological defense against viral infect … viral protein function. Co-transfection ISGylation analysis of SARS-CoV-2 proteins showed that the nucleocapsid (N) protein is ISGylated at several sites. Herc5 promoted N ISGylation and interacted with N, indicating that Herc5 acts as an E3 ligase for N ISGylation. Lys-261 (K261) within the oligomerization domain of N was identified as a potential ISGylation …

Моноклональная антитела для вирусного нуклеокапсида Рифт-Вэлли: применение в ИгГ/ИгМ ЭЛИСА для серодиагноза.

ВВЕДЕНИЕ: Вирус лихорадки Рифт-Вэлли (RVFV), принадлежащий семейству фенуивиридае, несет ответственность за зоонотическое заболевание под названием " лихорадка долины Рифт " . В настоящее время PVFV распространился от Африки до Азии, и в силу своей способности вызывать высокие показатели смертности он оказывает значительное воздействие на здоровье человека и экономическое развитие во многих обществах. Для клинического использования необходимы высокоспециализированные и чувствительные системы серодиагностики ВИЧ-инфекции. МЕТОД: BALB/c мыши были иммунизированы рекомбинантным нуклеокап

Совпадение в аннотации оригинала
INTRODUCTION: Rift Valley fever virus (RVFV) belonging to the Phenuiviridae family is responsible for a zoonotic disease called Rift Valley … FV infection are needed for clinical use. METHOD: BALB/c mice were immunized with recombinant RVFV nucleocapsid (rRVFV-N) protein and the spleen cells fused with SP2/0 myeloma cells to create hybridoma cell lines. The secreted monoclonal antibodies (MAbs) were purified and characterized. Enzyme-linked immunosorbent assay (ELISA) systems for the detection of I …

The preference signature of the SARS-CoV-2 Nucleocapsid NTD for its 5'-genomic RNA elements.

The nucleocapsid protein (N) of SARS-CoV-2 plays a pivotal role during the viral life cycle. It is involved in RNA transcription and accounts for packaging of the large genome into virus particles. N manages the enigmatic balance of bulk RNA-coating versus precise RNA-binding to designated cis-regulatory elements. Numerous studies report the involvement of its disordered segments in non-selective RNA-recognition, but how N organizes the inevitable recognition of specific motifs remains unanswered. We here use NMR spectroscopy to systematically analyze the interactions of N's N-terminal RNA-bin…

A highly sensitive nanobody-based immunoassay detecting SARS-CoV-2 nucleocapsid protein using all-recombinant reagents.

Antigen tests have been crucial for managing the COVID-19 pandemic by identifying individuals infected with SARS-CoV-2. This remains true even after immunity has been widely attained through natural infection and vaccination, since it only provides moderate protection against transmission and is highly permeable to the emergence of new virus variants. For this reason, the widespread availability of diagnostic methods is essential for health systems to manage outbreaks effectively. In this work, we generated nanobodies to the virus nucleocapsid protein (NP) and after an affinity-guided selectio…