Molecular characterization of SARS-CoV-2 nucleocapsid protein.

Corona Virus Disease 2019 (COVID-19) is a highly prevalent and potent infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Until now, the world is still endeavoring to develop new ways to diagnose and treat COVID-19. At present, the clinical prevention and treatment of COVID-19 mainly targets the spike protein on the surface of SRAS-CoV-2. However, with the continuous emergence of SARS-CoV-2 Variants of concern (VOC), targeting the spike protein therapy shows a high degree of limitation. The Nucleocapsid Protein (N protein) of SARS-CoV-2 is highly conserved in virus evolution and is involved in the key process of viral infection and assembly. It is the most expressed viral structural protein after SARS-CoV-2 infection in humans and has high immunogenicity. Therefore, N protein as the key factor of virus infection and replication in basic research and clinical application has great potential research value. This article reviews the research progress on the structure and biological function of SARS-CoV-2 N protein, the diagnosis and drug research of targeting N protein, in order to promote researchers' further understanding of SARS-CoV-2 N protein, and lay a theoretical foundation for the possible outbreak of new and sudden coronavirus infectious diseases in the future.

An indirect ELISA for detecting anti-SARS-CoV-2 antibodies in human sera using a baculovirus-expressed recombinant nucleocapsid antigen.

This study focuses on the development and initial assessment of an indirect IgG enzyme-linked immunosorbent assay (ELISA) specifically designed to detect of anti-SARS-CoV-2 antibodies. The unique aspect of this ELISA method lies in its utilization of a recombinant nucleocapsid (N) antigen, produced through baculovirus expression in insect cells. Our analysis involved 292 RT-qPCR confirmed positive serum samples and 54 pre-pandemic healthy controls. The process encompassed cloning, expression, and purification of the SARS-CoV-2 N gene in insect cells, with the resulted purified protein employed in our ELISA tests. Statistical analysis yielded an Area Under the Curve of 0.979, and the optimized cut-off exhibited 92 % sensitivity and 94 % specificity. These results highlight the ELISA's potential for robust and reliable serological detection of SARS-CoV-2 antibodies. Further assessments, including a larger panel size, reproducibility tests, and application in diverse populations, could enhance its utility as a valuable biotechnological solution for diseases surveillance.

Expression and purification of an NP-hoc fusion protein: Utilizing influenza a nucleoprotein and phage T4 hoc protein.

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.

Indiscriminate activities of different henipavirus polymerase complex proteins allow for efficient minigenome replication in hybrid systems.

The henipaviruses, including Nipah virus (NiV) and Hendra virus (HeV), are biosafety level 4 (BSL-4) zoonotic pathogens that cause severe neurological and respiratory disease in humans. To study the replication machinery of these viruses, we developed robust minigenome systems that can be safely used in BSL-2 conditions. The nucleocapsid (N), phosphoprotein (P), and large protein (L) of henipaviruses are critical elements of their replication machinery and thus essential support components of the minigenome systems. Here, we tested the effects of diverse combinations of the replication support proteins on the replication capacity of the NiV and HeV minigenomes by exchanging the helper plasmids coding for these proteins among the two viruses. We demonstrate that all combinations including one or more heterologous proteins were capable of replicating both the NiV and HeV minigenomes. Sequence alignment showed identities of 92% for the N protein, 67% for P, and 87% for L. Notably, variations in amino acid residues were not concentrated in the N-P and P-L interacting regions implying that dissimilarities in amino acid composition among NiV and HeV polymerase complex proteins may not impact their interactions. The observed indiscriminate activity of NiV and HeV polymerase complex proteins is different from related viruses, which can support the replication of heterologous genomes only when the whole polymerase complex belongs to the same virus. This newly observed promiscuous property of the henipavirus polymerase complex proteins likely attributed to their conserved interaction regions could potentially be harnessed to develop universal anti-henipavirus antivirals.IMPORTANCEGiven the severity of disease induced by Hendra and Nipah viruses in humans and the continuous emergence of new henipaviruses as well as henipa-like viruses, it is necessary to conduct a more comprehensive investigation of the biology of henipaviruses and their interaction with the host. The replication of henipaviruses and the development of antiviral agents can be studied in systems that allow experiments to be performed under biosafety level 2 conditions. Here, we developed robust minigenome systems for the Nipah virus (NiV) and Hendra virus (HeV) that provide a convenient alternative for studying NiV and HeV replication. Using these systems, we demonstrate that any combination of the three polymerase complex proteins of NiV and HeV could effectively initiate the replication of both viral minigenomes, which suggests that the interaction regions of the polymerase complex proteins could be effective targets for universal and effective anti-henipavirus interventions.

[Prokaryotic expression of N protein of akabane disease virus and preparation of monoclonal antibody].

In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein. Following purification, BALB/c mice were immunized, and their splenocytes were fused with mouse myeloma cells (SP2/0) to produce hybridoma cells. The indirect ELISA method was used to screen for positive hybridoma cells. Two specific hybridoma cell lines targeting AKAV N protein, designated as 2C9 and 5E9, were isolated after three rounds of subcloning. Further characterization was conducted through ELISA, Western blotting, and indirect immunofluorescence assay (IFA). The results confirmed that the monoclonal antibodies specifically target AKAV N protein, exhibiting strong reactivity in IFA. Subtype analysis identified the heavy chain of the 2C9 mAb's as IgG2b and its light chain as κ-type; the 5E9 mAb's heavy chain was determined to be IgG1, with a κ-type light chain. Their ELISA titers reached 1:4 096 000. This study successfully developed two monoclonal antibodies targeting AKAV N protein, which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control, as well as for studying the function of the AKAV N protein.

Identification of B-cell epitope on the N protein of type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using monoclonal antibody and construction of epitope-mutated virus.

The escalating epidemic of PRRSV-1 in China has prompted widespread concern regarding the evolution of strains, disparities in pathogenicity to herds, and immunological detection of emerging strains. The nucleocapsid (N) protein, as a highly conserved protein with immunogenic properties in PRRSV, is a subject of intensive study. In this research, the recombinant His-N protein was expressed based on the N gene of PRRSV-1 using a prokaryotic expression system and then administered to BALB/c mice. A cell fusion protocol was implemented between SP2/0 cells and splenocytes, resulting in the successful screening of a monoclonal antibody against the N protein, designated as mAb 2D7, by indirect ELISA. Western Blot analysis and Indirect Immunofluorescence Assay (IFA) confirmed that mAb 2D7 positively responded to PRRSV-1. By constructing and expressing a series of truncated His-fused N proteins, a B-cell epitope of N protein, 59-AAEDDIR-65, was identified. A sequence alignment of two genotypes of PRRSV revealed that this epitope is relatively conserved in PRRSV, yet more so in genotype 1. Cross-reactivity analysis by Western blot analysis demonstrated that the B-cell epitope containing D62Y mutation could not be recognized by mAb 2D7. The inability of mAb 2D7 to recognize the epitope carrying the D62Y mutation was further determined using an infectious clone of PRRSV. This research may shed light on the biological significance of the N protein of PRRSV, paving the way for the advancement of immunological detection and development of future recombinant marker vaccine.

Insertion of foreign genes into the simian varicella virus genome by Tn7-mediated site-specific transposition.

A Tn7-transposition approach was utilized for site-specific insertion of foreign genes into the genome of simian varicella virus (SVV), the causative agent of simian varicella in nonhuman primates. The severe acute respiratory syndrome coronavirus (SARS-CoV-2) nucleocapsid (N) gene and receptor binding domain (RBD) of the spike gene were inserted into the ORF 14 region of the SVV genome cloned into a bacterial artificial chromosome and then transfected into Vero cells to generate the infectious recombinant SVV (rSVV). The rSVV replicated efficiently in infected Vero cells and expressed the N and RBD antigens as indicated by immunoblot and immunofluorescence assays. Tn7-mediated transposition provides a rapid and efficient method for constructing rSVVs which may be evaluated as live-attenuated vaccines.

Development and application of a blocking ELISA based on a N protein monoclonal antibody for the antibody detection against porcine reproductive and respiratory syndrome virus 2.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most widespread illnesses in the world's swine business. To detect the antibodies against PRRSV-2, a blocking enzyme-linked immunosorbent assay (B-ELISA) was developed, utilizing a PRRSV-2 N protein monoclonal antibody as the detection antibody. A checkerboard titration test was used to determine the optimal detection antibody dilution, tested pig serum dilution and purified PRRSV coated antigen concentration. After analyzing 174 negative pig sera and 451 positive pig sera, a cutoff value of 40 % was selected to distinguish between positive and negative sera using receiver operating characteristic curve analysis. The specificity and sensitivity of the assay were evaluated to equal 99.8 % and 96 %, respectively. The method had no cross-reaction with PCV2, PRV, PPV, CSFV, PEDV, TGEV, and PRRSV-1 serum antibodies, and the coefficients of variation of intra-batch and inter-batch repeatability experiments were both <10 %. A total of 215 clinical serum samples were tested, and the relative coincidence rate with commercial ELISA kit was 99.06 %, and the kappa value was 0.989, indicating that these two detection results exhibited high consistency. Overall, the B-ELISA should serve as an ideal method for large-scale serological investigation of PRRSV-2 antibodies in domestic pigs.

Modular design of bi- and multi-specific knob domain fusions.

Introduction: The therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively. Methods: Autonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights. Results: Knob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference. Discussion: Compared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides.

Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibiting TRIM25 oligomerization and functional activation of RIG-I/TRIM25.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), an emerging Alpha-coronavirus, brings huge economic loss in swine industry. Interferons (IFNs) participate in a frontline antiviral defense mechanism triggering the activation of numerous downstream antiviral genes. Here, we demonstrated that TRIM25 overexpression significantly inhibited SADS-CoV replication, whereas TRIM25 deficiency markedly increased viral yield. We found that SADS-CoV N protein suppressed interferon-beta (IFN-ß) production induced by Sendai virus (SeV) or poly(I:C). Moreover, we determined that SADS-CoV N protein interacted with RIG-I N-terminal two caspase activation and recruitment domains (2CARDs) and TRIM25 coiled-coil dimerization (CCD) domain. The interaction of SADS-CoV N protein with RIG-I and TRIM25 caused TRIM25 multimerization inhibition, the RIG-I-TRIM25 interaction disruption, and consequent the IRF3 and TBK1 phosphorylation impediment. Overexpression of SADS-CoV N protein facilitated the replication of VSV-GFP by suppressing IFN-ß production. Our results demonstrate that SADS-CoV N suppresses the host IFN response, thus highlighting the significant involvement of TRIM25 in regulating antiviral immune defenses.

How does severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) achieve immune evasion?: A narrative review.

COVID-19 caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2, (SARS-CoV-2) is a highly contagious disease known for its significant lung damage. Although the impact of the COVID-19 pandemic on our daily lives has been limited, the virus has not vanished entirely and continues to undergo mutations. This calls for a concentrated focus on the matter of SARS-CoV-2 immune evasion. Drawing on observations of immune escape mechanisms in other viruses, some scholars have proposed that liquid-liquid phase separation might play a crucial role in SARS-CoV-2's ability to evade the immune system. Within the structure of SARS-CoV-2, the nucleocapsid protein plays a pivotal role in RNA replication and transcription. Concurrently, this protein can engage in phase separation with RNA. A thorough examination of the phase separation related to the nucleocapsid protein may unveil the mechanism by which SARS-CoV-2 accomplishes immune evasion. Moreover, this analysis may provide valuable insights for future development of innovative antiviral drugs or vaccines.

The immune-evasive proline-283 substitution in influenza nucleoprotein increases aggregation propensity without altering the native structure.

Nucleoprotein (NP) is a key structural protein of influenza ribonucleoprotein complexes and is central to viral RNA packing and trafficking. NP also determines the sensitivity of influenza to myxovirus resistance protein 1 (MxA), an innate immunity factor that restricts influenza replication. A few critical MxA-resistant mutations have been identified in NP, including the highly conserved proline-283 substitution. This essential proline-283 substitution impairs influenza growth, a fitness defect that becomes particularly prominent at febrile temperature (39°C) when host chaperones are depleted. Here, we biophysically characterize proline-283 NP and serine-283 NP to test whether the fitness defect is caused by the proline-283 substitution introducing folding defects. We show that the proline-283 substitution changes the folding pathway of NP, making NP more aggregation prone during folding, but does not alter the native structure of the protein. These findings suggest that influenza has evolved to hijack host chaperones to promote the folding of otherwise biophysically incompetent viral proteins that enable innate immune system escape.

A novel linear B cell epitope of the canine coronavirus nucleocapsid protein identified by a monoclonal antibody.

The infection of canine coronavirus (CCoV) causes a highly contagious disease in dogs with acute gastroenteritis. The efficient serological diagnostics is critical for controlling the disease caused by CCoV. Nucleocapsid (N) protein of CCoV is an important target for developing serological approaches. However, little is known about the antigenic sites in the N protein of CCoV. In this study, we generated a monoclonal antibody (mAb) against the N protein of CCoV, designated as 13E8, through the fusion of the sp2/0 cells with the spleen cells from a mouse immunized with the purified recombinant GST-N protein. Epitope mapping revealed that mAb 13E8 recognized a novel linear B cell epitope in N protein at 294-314aa (named as EP-13E8) by using a serial of truncated N protein through Western blot and ELISA. Sequence analysis showed that the sequence of EP-13E8 was highly conserved (100 %) among different CCoV strains analyzed, but exhibited a low similarity (31.8-63.6 %) with the responding sequence in other coronaviruses of the same genus such as FCoV, PEDV and HCoV except for TGEV (95.5 % identity). Structural assay suggested that the epitope of EP-13E8 were located in the close proximity on the surface of the N protein. Overall, the mAb 13E8 against N protein generated and its epitope EP-13E8 identified here paid the way for further developing epitope-based serological diagnostics for CCoV.

Two Phylogenetic Cohorts of the Nucleocapsid Protein NP and Their Correlation with the Host Range of Influenza A Viruses.

Influenza A virus has a wide natural areal among birds, mammals, and humans. One of the main regulatory adaptors of the virus host range is the major NP protein of the viral nucleocapsid. Phylogenetic analysis of the NP protein of different viruses has revealed the existence of two phylogenetic cohorts in human influenza virus population. Cohort I includes classical human viruses that caused epidemics in 1957, 1968, 1977. Cohort II includes the H1N1/2009pdm virus, which had a mixed avian-swine origin but caused global human pandemic. Also, the highly virulent H5N1 avian influenza virus emerged in 2021 and caused outbreaks of lethal infections in mammals including humans, appeared to have the NP gene of the second phylogenetic cohort and, therefore, by the type of adaptation to human is similar to the H1N1/2009pdm virus and seems to possess a high epidemic potential for humans. The data obtained shed light on pathways and dynamics of adaptation of avian influenza viruses to humans and propose phylogenetic algorithm for systemic monitoring of dangerous virus strains to predict epidemic harbingers and take immediate preventive measures.

The nucleocapsid protein facilitates p53 ubiquitination-dependent proteasomal degradation via recruiting host ubiquitin ligase COP1 in PEDV infection.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of the coronavirus family and caused severe economic losses to the global swine industry. Previous studies have established that p53 is a host restriction factor for PEDV infection, and p53 degradation occurs in PEDV-infected cells. However, the underlying molecular mechanisms through which PEDV viral proteins regulate p53 degradation remain unclear. In this study, we found that PEDV infection or expression of the nucleocapsid protein downregulates p53 through a post-translational mechanism: increasing the ubiquitination of p53 and preventing its nuclear translocation. We also show that the PEDV N protein functions by recruiting the E3 ubiquitin ligase COP1 and suppressing COP1 self-ubiquitination and protein degradation, thereby augmenting COP1-mediated degradation of p53. Additionally, COP1 knockdown compromises N-mediated p53 degradation. Functional mapping using truncation analysis showed that the N-terminal domains of N protein were responsible for interacting with COP1 and critical for COP1 stability and p53 degradation. The results presented here suggest the COP1-dependent mechanism for PEDV N protein to abolish p53 activity. This study significantly increases our understanding of PEDV in antagonizing the host antiviral factor p53 and will help initiate novel antiviral strategies against PEDV.

Functionality of IAV packaging signals depends on site-specific charges within the viral nucleoprotein.

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.

Vimentin inhibits peste des petits ruminants virus replication by interaction with nucleocapsid protein.

The Peste des petits ruminant virus (PPRV) is a member of the Paramyxoviridae family and is classified into the genus Measles virus. PPRV predominantly infects small ruminants, leading to mortality rates of nearly 100%, which have caused significant economic losses in developing countries. Host proteins are important in virus replication, but the PPRV nucleocapsid (N) protein-host interacting partners for regulating PPRV replication remain unclear. The present study confirmed the interaction between PPRV-N and the host protein vimentin by co-immunoprecipitation and co-localization experiments. Overexpression of vimentin suppressed PPRV replication, whereas vimentin knockdown had the opposite effect. Mechanistically, N was subjected to degradation via the ubiquitin/proteasome pathway, where vimentin recruits the E3 ubiquitin ligase NEDD4L to fulfill N-ubiquitination, resulting in the degradation of the N protein. These findings suggest that the host protein vimentin and E3 ubiquitin ligase NEDD4L have an anti-PPRV effect.

Impact of mutations on the stability of SARS-CoV-2 nucleocapsid protein structure.

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․.

Ultrasensitive Detection of SARS-CoV-2 Nucleocapsid Protein Based on Porphyrin-Based Metal-Organic Gels with Highly Efficient Electrochemiluminescence at Low Potential.

Metal-organic gels (MOGs) are a new type of intelligent soft material, which are bridged by metal ions and organic ligands through noncovalent interactions. In this paper, we prepared highly stable P-MOGs, using the classical organic electrochemiluminescence (ECL) luminescence meso-tetra(4-carboxyphenyl)porphine as the organic ligand and Fe3+ as the metal ion. Surprisingly, P-MOGs can stably output ECL signals at a low potential. We introduced P-MOGs into the ECL resonance energy transfer strategy (ECL-RET) and constructed a quenched ECL immunosensor for the detection of the SARS-CoV-2 nucleocapsid protein (SARS-CoV-2-N). In the ECL-RET system, P-MOGs were used as energy donors, and Au@Cu2O@Fe3O4 were selected as energy acceptors. The ultraviolet-visible spectrum of Au@Cu2O@Fe3O4 partially overlaps with the ECL spectrum of P-MOGs, which can effectively touch off the ECL-RET behavior between the donors and receptors. Under the ideal experimental situation, the linear detection range of the SARS-CoV-2-N concentration was 10 fg/mL to 100 ng/mL, and the limit of detection was 1.5 fg/mL. This work has broad application prospects for porphyrin-MOGs in ECL sensing.