• KSBB Journal
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• v.13 no.6
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• pp.656-661
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• 1998

Hantaan virus belonging to the genus Hantavirus and family Bunyaviridae causes an acute severe illness of human, Haemorrhagic Fever with Renal Syndrome (HFRS). It is a rodent host-borne pathogen and distributed in Asia and Eastern Europe. Hantaviruses have three major antigens, i.e., G1, G2 glycoproteins and nucleocapsid protein (N). Among them, nucleocapsid protein was reported to be the most invaluable antigen as for diagnosis. We have cloned and expressed Hantaan viral nucleocapsid gene in E. coli BL21(DE3). In this study, we have tried to purify the nucleocapsid protein produced by recombinant E. coli, and could attained a purity of >90% by anti-N monoclonal antibody-coupled immunoaffinity chromatography or phenyl sepharose hydrophobic interaction chromatography.

• Journal of Microbiology
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• v.44 no.5
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• pp.577-580
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• 2006

GST pull-down assays were used to characterize the SARS-CoV membrane (M) and nucleocapsid (N) interaction, and it was found that the amino acids 211-254 of N protein were essential for this interaction. When tetrad glutamines (Q) were replaced with glutamic acids (E) at positions of 240-243 of the N protein, the interaction was disrupted.

• Korean Journal of Veterinary Research
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• v.43 no.1
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• pp.129-137
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• 2003

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failures in sows and respiratory problems in piglets. The nucleocapsid(N) protein, encoded by the open reading frame 7 (ORF7) gene, is known to be the most abundant and antigenic protein in PRRS virus. Therefore, it was suggested that the N protein could be a suitable candidate for the detection of PRRS virus-specific antibodies and diagnosis of PRRS. In the present study, the ORF7 gene encoding the N protein was cloned and expressed as a fusion protein with the glutathione S-transferase (GST) in Escherichia coli. The resulting GST-N recombinant protein was used as an antigen for an indirect sandwich enzyme-linked immunosorbent assay (i-ELISA). Expressed GST-N recombinant protein was migrated at 41 kDa and reacted with ORF7-specific monoclonal antibody by Western blotting. In order to increase the specificity of the ELISA for the detection of PRRS virus-specific antibodes, an i-ELISA was developed using an anti-GST antibody as a capture antibody. The sensitivity and specificity of developed i-ELISA were 92% and 96%, respectively. Based on these results, it was suggested that the i-ELISA is a simple and rapid test for screening a large number of swine sera for the anti-PRRS virus antibodies.

• Microbiology and Biotechnology Letters
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• v.37 no.3
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• pp.287-292
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• 2009

The nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is a basic multifunctional protein which has been reported to be a serine phosphoprotein with yet-identified functions. As a first step towards understanding the general role of N protein phosphorylation during virus replication, the non-phosphorylated mutant N gene was constructed by mutating all serine residues to alanine. This recombinant N protein was identified to be unphosphorylated, confirming that serine residues truly function as core amino acids responsible for N protein phosphorylation. The PRRSV N protein has been shown to possess the biological features of nuclear localization and N-N homodimerization which individually play critical roles in virus infection. In the present study, therefore, it was attempted to investigate whether these two properties of the N protein are modulated by its phosphorylation status. However, experimental results showed that the non-phosphorylated N protein was still present in the nucleus and nucleolus, and was able to associate with itself by non-covalent interactions. Taken together, the data suggest phosphorylation-independent regulation of N protein nuclear transport or oligomerization, thereby implying the potential involvement of phosphorylation in regulating the activities of the N protein at other levels including RNA-binding capacity.

• Biomolecules & Therapeutics
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• v.30 no.5
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• pp.427-434
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• 2022

The drug repurposing strategy has been applied to the development of emergency COVID-19 therapeutic medicines. Current drug repurposing approaches have been directed against RNA polymerases and viral proteases. Recently, we found that the inhibition of the interaction between the SARS-CoV-2 structural nucleocapsid (N) and spike (S) proteins decreased viral replication. In this study, drug repurposing candidates were screened by in silico molecular docking simulation with the SARS-CoV-2 structural N protein. In the ChEMBL database, 1994 FDA-approved drugs were selected for the in silico virtual screening against the N terminal domain (NTD) of the SARS-CoV-2 N protein. The tyrosine 109 residue in the NTD of the N protein was used as the center of the ligand binding grid for the docking simulation. In plaque forming assays performed with SARS-CoV-2 infected Vero E6 cells, atovaquone, abiraterone acetate, and digoxin exhibited a tendency to reduce the size of the viral plagues without affecting the plaque numbers. Abiraterone acetate significantly decreased the accumulation of viral particles in the cell culture supernatants in a concentration-dependent manner. In addition, abiraterone acetate significantly decreased the production of N protein and S protein in the SARS-CoV-2-infected Vero E6 cells. In conclusion, abiraterone acetate has therapeutic potential to inhibit the viral replication of SARS-CoV-2.

• KSBB Journal
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• v.13 no.6
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• pp.662-668
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• 1998

Viruses belonging to the Hantavirus genus cause two acute severe illness in humans, i.e., Haemorrhagic Fever with Renal Syndrome (HFRS) and Hantavirus Pulmonary Syndrome(HPS). Among them, Hantaan virus is one of the most important viruses causing HFRS. Recombinant expression vectors, pKK-NP and pET-NP, with Hantaan viral nucleocapsid gene were constructed, and used to transform Eschericia coli BL21(DE3). Stability of the vectors in the host strain, and effects of some environmental conditions on the expression of nucleocapsid gene were studied. Expression vector, pKK-NP, was very unstable, and the expression level of nucleocapsid gene was very low compared to that of pET-NP. BL21(pET-NP) produced about 100 mg of N protein per liter of culture broth. Induction time did not show any significant difference on the expression level of nucleocapsid gen and cell growth. BL21(pET-NP) culture at 35$^{\circ}C$ showed a little higher expression level than at 30$^{\circ}C$ during growth phase, but reached to the same level at stationary phase. Total expression level was proportional to supplemented glucose concentration of media up to 0.5% along with cell growth, but expression level per unit cell mass was inversely proportional to glucose concentration and maximal when glucose was not supplemented at all.

• Genomics & Informatics
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• v.18 no.4
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• pp.43.1-43.13
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• 2020

The coronavirus disease 2019 is a contagious disease and had caused havoc throughout the world by creating widespread mortality and morbidity. The unavailability of vaccines and proper antiviral drugs encourages the researchers to identify potential antiviral drugs to be used against the virus. The presence of RNA binding domain in the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be a potential drug target, which serves multiple critical functions during the viral life cycle, especially the viral replication. Since vaccine development might take some time, the identification of a drug compound targeting viral replication might offer a solution for treatment. The study analyzed the phylogenetic relationship of N protein sequence divergence with other 49 coronavirus species and also identified the conserved regions according to protein families through conserved domain search. Good structural binding affinities of a few natural and/or synthetic phytocompounds or drugs against N protein were determined using the molecular docking approaches. The analyzed compounds presented the higher numbers of hydrogen bonds of selected chemicals supporting the drug-ability of these compounds. Among them, the established antiviral drug glycyrrhizic acid and the phytochemical theaflavin can be considered as possible drug compounds against target N protein of SARS-CoV-2 as they showed lower binding affinities. The findings of this study might lead to the development of a drug for the SARS-CoV-2 mediated disease and offer solution to treatment of SARS-CoV-2 infection.

• BMB Reports
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• v.38 no.4
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• pp.381-385
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• 2005

Severe acute respiratory syndrome (SARS) is an emerging infectious disease associated with a novel coronavirus (CoV) that was identified and molecularly characterized in 2003. Previous studies on various coronaviruses indicate that protein-protein interactions amongst various coronavirus proteins are critical for viral assembly and morphogenesis. It is necessary to elucidate the molecular mechanism of SARS-CoV replication and rationalize the anti-SARS therapeutic intervention. In this study, we employed an in vitro GST pull-down assay to investigate the interaction between the membrane (M) and the nucleocapsid (N) proteins. Our results show that the interaction between the M and N proteins does take place in vitro. Moreover, we provide an evidence that 12 amino acids domain (194-205) in the M protein is responsible for binding to N protein. Our work will help shed light on the molecular mechanism of the virus assembly and provide valuable information pertaining to rationalization of future anti-viral strategies.

• Molecules and Cells
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• v.45 no.12
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• pp.911-922
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• 2022

A structural protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), nucleocapsid (N) protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3β constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), found in many endogenous GSK-3β binding proteins, such as Axins, FRATs, WWOX, and GSKIP. Indeed, N interacts with GSK-3β similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. The N phosphorylation is also required for its structural loading in a virus-like particle (VLP). Compared to other coronaviruses, N of Sarbecovirus lineage including bat RaTG13 harbors a CDK1-primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3β. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allow increased abundance and hyper-phosphorylation of N. Our observations suggest that GID and mutations for increased phosphorylation in N may have contributed to the evolution of variants.

• BMB Reports
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• v.38 no.3
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• pp.354-359
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• 2005

Open reading frame 29 (ha29) is a gene specific for Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearSNPV). Sequence analyses showed that the transcription factor Tfb2 motif, bromodomain and Half-A-TPR (HAT) repeat were present at aa 66-82, 4-76, 55-90 of the Ha29 protein respectively. The product of Ha29 was detected in HearSNPV-infected HzAM1 cells at 3 h post-infection. Western blot analysis using a polyclonal antibody produced by immunizing a rabbit with purified GST-Ha29 fusion protein indicates that Ha29 is an early gene. The size of Ha29 product in infected HzAM1 cells was about 25 kDa, which was larger than the presumed size of 20.4 kDa. Tunicamycin treatment of HearSNPV-infected HzAM1 cells suggested that the Ha29 protein is N-glycosylated. Fluorescent confocal laser scanning microscope examination, and Western blot analysis of purified budded virus (BVs), occlusion-derived virus (ODVs), cell nuclear and cytoplasmic fraction, showed that the Ha29 protein was localized in the nucleus. Our results suggested that ha29 of HearSNPV encodes a non-structurally functional protein that may be associated with virus gene transcription in Helicoverpa hosts.