A versatile building block:the structures and functions of negative-sense single-stranded RNA virus nucleocapsid proteins

Nucleocapsid protein(NPs)of negative-sense single-stranded RNA(-ssRNA)viruses function in different stages of viral replication,transcription,and maturation.Structural investigations show that-ssRNA viruses that encode NPs preliminarily serve as structural building blocks that encapsidate and protect the viral genomic RNA and mediate the interaction between genomic RNA and RNA-dependent RNA polymerase.However,recent structural results have revealed other bio-logical functions of-ssRNA viruses that extend our understanding of the versatile roles of virally encoded NPs.

Ciclopirox inhibits SARS-CoV-2 replication by promoting the degradation of the nucleocapsid protein

The nucleocapsid protein(NP)plays a crucial role in SARS-CoV-2 replication and is the most abundant structural protein with a long half-life.Despite its vital role in severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)assembly and host inflammatory response,it remains an unexplored target for drug development.In this study,we identified a small-molecule compound(ciclopirox)that promotes NP degradation using an FDA-approved library and a drug-screening cell model.Ciclopirox significantly inhibited SARS-CoV-2 replication both in vitro and in vivo by inducing NP degradation.Ciclopirox induced abnormal NP aggregation through indirect interaction,leading to the formation of condensates with higher viscosity and lower mobility.These condensates were subsequently degraded via the autophagy-lysosomal pathway,ultimately resulting in a shortened NP half-life and reduced NP expression.Our results suggest that NP is a potential drug target,and that ciclopirox holds substantial promise for further development to combat SARS-CoV-2 replication.

Analysis of the N Protein Sequence Variability in 13 Isolated PRRSV Strains from China

Object: To analyze porcine reproductive and respiratory syndrome virus (PRRSV) strains from 13 infection cases via the N protein gene and its encoded amino acid sequence and to provide a theoretical basis for the epidemiological study, prevention and control of porcine reproductive and respiratory syndrome (PRRS). Methods: In clinically suspected PRRSV infections, viruses were isolated by extracting viral nucleic acid and amplifying the N protein gene by RT-PCR. Then, the product was purified and sequenced to acquire the whole gene sequence of the N protein and its encoded amino acid sequence. DNASTAR software was used to analyze the homology, the genetic evolution and the derivation of the variability of amino acids of the N protein gene from 13 PRRSV strains and classical domestic and foreign strains. Results: Among the thirteen strains of PRRSV isolated from this study, ten strains had the greatest homology with the JXA1 strain (98.9% - 100%), and they belonged to the sublineage 8.7. The remaining three strains had the greatest homology with the NADC30 strain (95.4% - 97.1%), and they belonged to lineage one. The analysis of the variability of N protein amino acids showed that there were high frequency mutations in the five loci of 13 isolated strains of PRRSV as follows: 15th amino acid (10/13), 46th amino acid (11/13), 91st amino acid (10/13), 109th amino acid (10/13), and 117th amino acid (10/13). Conclusion: In recent years, sublineage 8.7 was the dominant pedigree in field PRRSV epidemic strains in China with lineage one occupying a certain proportion of the field. Four high frequency mutations existed in N protein antigen epitopes of isolated strains from the region. The nuclear localization signal (NLS) structure, specifically the 46th amino acid residue of the N protein, was mutated and genetically stable.

Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

The outbreak of coronavirus disease(COVID-19)caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths.Currently,there is no specific viral protein-targeted therapeutics.Viral nucleocapsid protein is a potential antiviral drug target,serving multiple critical functions during the viral life cycle.However,the structural information of SARS-CoV-2 nucleocapsid protein remains unclear.Herein,we have determined the 2.7 A crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein.Although the overall structure is similar as other reported coronavirus nucleocapsid protein N-terminal domain,the surface electrostatic potential characteristics between them are distinct.Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside theβ-sheet core.Complemented by in vitro binding studies,our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain,guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

The Epitope Study on the SARS-CoV Nucleocapsid Protein

The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this protein. A detailed screening of peptide with ELISA demonstrated that the amino sequence from Codons 371 to 407 was the epitope site at the C-terminus of the N protein. Understanding of the epitope sites could be very significant for developing an effective diagnostic approach to SARS.

Comparative Immunization in BALB/c Mice with Recombinant Replication-Defective Adenovirus Vector and DNA Plasmid Expressing a SARS-CoV Nucleocapsid Protein Gene

In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus （SARS-CoV）-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ （IFN-γ） secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd-N was significantly stronger than that of pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific （IFN-γ） secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirns boosting could be used as a potential SARS-CoV vaccine.

The C-Terminal Portion of the Nucleocapsid Protein Demonstrates SARS-CoV Antigenicity

In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

The nucleocapsid protein of rice stripe virus in cell nuclei of vector insect regulates viral replication

Rice stripe virus(RSV)transmitted by the small brown planthopper causes severe rice yield losses in Asian countries.Although viral nuclear entry promotes viral replication in host cells,whether this phenomenon occurs in vector cells remains unknown.Therefore,in this study,we systematically evaluated the presence and roles of RSV in the nuclei of vector insect cells.We observed that the nucleocapsid protein(NP)and viral genomic RNAs were partially transported into vector cell nuclei by utilizing the importin a nuclear transport system.When blocking NP nuclear localization,cytoplasmic RSV accumulation significantly increased.In the vector cell nuclei,NP bound the transcription factor YY1 and affected its positive regulation to FAIM.Subsequently,decreased FAIM expression triggered an antiviral caspase-dependent apoptotic reaction.Our results reveal that viral nuclear entry induces completely different immune effects in vector and host cells,providing new insights into the balance between viral load and the immunity pressure in vector insects.

Structures of the N- and C-terminal domains of MHV-A59 nucleocapsid protein corroborate a conserved RNA-protein binding mechanism in coronavirus

Coronaviruses are the causative agent of respiratory and enteric diseases in animals and humans.One example is SARS,which caused a worldwide health threat in 2003.In coronaviruses,the structural protein N(nucleocapsid protein)associates with the viral RNA to form the filamentous nucleocapsid and plays a crucial role in genome replication and transcription.The structure of Nterminal domain of MHV N protein also implicated its specific affinity with transcriptional regulatory sequence(TRS)RNA.Here we report the crystal structures of the two proteolytically resistant N-(NTD)and C-terminal(CTD)domains of the N protein from murine hepatitis virus(MHV).The structure of NTD in two different crystal forms was solved to 1.5Å.The higher resolution provides more detailed structural information than previous reports,showing that the NTD structure from MHV shares a similar overall and topology structure with that of SARS-CoV and IBV,but varies in its potential surface,which indicates a possible difference in RNA-binding module.The structure of CTD was solved to 2.0-Åresolution and revealed a tightly intertwined dimer.This is consistent with analytical ultracentrifugation experiments,suggesting a dimeric assembly of the N protein.The similarity between the structures of these two domains from SARS-CoV,IBV and MHV corroborates a conserved mechanism of nucleocapsid formation for coronaviruses.

Mass Spectrometry Analysis of SARS-CoV-2 Nucleocapsid Protein Reveals Camouflaging Glycans and Unique Post-Translational Modifications

The devastating coronavirus disease 2019(COVID-19)pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and its viral components.Compared to the Spike protein,which is the primary target for currently available vaccines or antibodies,knowledge about other virion structural components is incomplete.Using high-resolution mass spectrometry,we report a comprehensive post-translational modification(PTM)analysis of nucleocapsid phosphoprotein(NCP),the most abundant structural component of the SARS-CoV-2 virion.In addition to phosphoryl groups,we show that the SARS-CoV-2 NCP is decorated with a variety of PTMs,including N-glycans and ubiquitin.Based on newly identified PTMs,refined protein structural models of SARS-CoV-2 NCP were proposed and potential immune recognition epitopes of NCP were aligned with PTMs.These data can facilitate the design of novel vaccines or therapeutics targeting NCP,as valuable alternatives to the current vaccination and treatment paradigm that is under threat of the ever-mutating SARS-CoV-2 Spike protein.

Antigenicity of severe fever with thrombocytopenia syndrome virus nucleocapsid protein and its potential application in the virus serodiagnosis

Dear Editor,Severe fever with thrombocytopenia syndrome virus(SFTSV)is a newly identified viral pathogen of the genus Phlebovirus in the family Bunyaviridae(Sun et al.,2012).SFTSV was first identified from patient serum samples in China(Li et al.,2013;Ning et al.,2015).SFTSV can cause a severe hemorrhagic fever-like disease with a reported case fatality rate ranging from 2.5%

Enhancing DNA vaccine potency against hantavirus by co-administration of interleukin-12 expression vector as a genetic adjuvant

Background The heavy incidence and mortality of hemorrhagic fever with renal syndrome, as well as no specific drugs in curing the disease, clearly indicate the need for development Of the more effective hantavirus vaccine. Refining the DNA vaccination strategy to elicit more clinically efficacious immune responses is now under intensive investigation. In the present study, we examined the effects of using an interleukin-12 expression plasmid as a genetic adjuvant to enhance the immune responses induced by a DNA vaccine based on the S gene encoding nucleocapsid protein against hantavirus. Methods BALB/c mice were immunized three times by intramuscular inoculations of DNA vaccine encoding of hantanvirus nucleoeapsid protein alone or in combination with a plasmid expressing murine interleukin-12 (pcIL-12). Booster immunizations were employed 2 times at 2-week interval. To evaluate the Immoral and cellular immune responses, antigen-specific lymphocyte proliferation and antibody production were assayed by MTT method and ELISA respectively. The level of interleukin-4 and interferon-gamma in the splenic lymphocytic cultured supernatant were detected with ELISA kit at day 5, 10, 17, 35 and 42 after primary immunization. Results Antigen-specific IgG antibodies was increased markedly at day 17 in the experiment groups and reached a plateau after day 35. As pcIL-12 co-injected, a significant inhibition of antigen-specific IgG levels was displayed over the period and the antibody mean titre was decreased to only about 1: 50 at day 42 after primary immunization, significantly lower than the group immunized with pcDNA3.1 + S alone, in which the mean titre was about 1:70. Interferon-gamma was increased remarkably by the co-injection of pcIL-12 compared with the injection of pcDNA3.1 + S alone. However, the production of interleukin-4 was inhibited by pcIL-12 coinjection. Furthermore, pcIL-12 co-injection efficiently enhanced antigen-specific lymphocyte proliferation. Conclusion Humoral and cytokine responses elicited by pcDNA3.1 + S inoculation can be modulated by coinoculation with pcIL-12 and efficiently induced Th1-dominant immune responses.

SARS-CoV-2 impairs the disassembly of stress granules and promotes ALS-associated amyloid aggregation

The nucleocapsid(N)protein of SARS-CoV-2 has been reported to have a high ability of liquid-liquid phase separation,which enables its incorporation into stress granules(SGs)of host cells.However,whether SG invasion by N protein occurs in the scenario of SARS-CoV-2 infection is unknow,neither do we know its con-sequence.Here,we used SARS-CoV-2 to infect mam-malian cells and observed the incorporation of N protein into SGs,which resulted in markedly impaired self-dis-assembly but stimulated cell cellular clearance of SGs.NMR experiments further showed that N protein binds to the SG-related amyloid proteins via non-specific tran-sient interactions,which not only expedites the phase transition of these proteins to aberrant amyloid aggre-gation in vitro,but also promotes the aggregation of FUS with ALS-associated P525L mutation in cells.In addition,we found that ACE2 is not necessary for the infection of SARS-CoV-2 to mammalian cells.Our work indicates that SARS-CoV-2 infection can impair the dis-assembly of host SGs and promote the aggregation of SG-related amyloid proteins,which may lead to an increased risk of neurodegeneration.