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Lee Beomki,Ko Jae-Hoon,Baek Jin Yang,Kim Haein,Huh Kyungmin,Cho Sun Young,Kang Cheol-In,Chung Doo Ryeon,Peck Kyong Ran,Kang Eun-Suk 대한의학회 2023 Journal of Korean medical science Vol.38 No.37
As nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 is immunogenic but not targeted in vaccines, it could be useful in distinguishing natural infection from vaccination. We aimed to investigate the clinical utility of sero-immunological responses against the nucleocapsid protein. Nucleocapsid antibody immunoassay study with 302 coronavirus disease 2019 (COVID-19) patients showed lower titers in immunocompromised patients (P < 0.001), higher titers in higher severity (P = 0.031), and different seroconversion rates and titers according to variants of concern. Longitudinal evaluation of nucleocapsid antibodies using 513 samples from 291 COVID-19 patients revealed that it could persist up to 556 days from symptom onset. Interferon gamma release assay against the nucleocapsid protein showed poor response, precluding the deduction of a cut-off for the nucleocapsid protein. In conclusion, nucleocapsid antibody provides instructive clues about the immunogenicity of nucleocapsid proteins by different seroconversion rates and titers according to the severity of infection, host immune status, and different variants of concern.
Fang, Xiaonan,Ye, Linbai,Timani, Khalid Amine,Li, Shanshan,Zen, Yingchun,Zhao, Meng,Zheng, Hong,Wu, Zhenghui Korean Society for Biochemistry and Molecular Biol 2005 Journal of biochemistry and molecular biology Vol.38 No.4
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.
임대준,박범석,최궤문,강석권 한국곤충학회 1989 Korean journal of entomology Vol.19 No.2
담배거세미나방 핵다각체병 바이러스의 단백질을 SDS-PAGE한 결과, 다각체 단백질은 분자양 31 Kd.의 major band와 7개의 minor band로 분리되었으며, 비리온 단백질은 27개의 band로 관찰되었고, 그중 비교적 작은 분자량의 12개 band는 뉴클레오캡시드 단백질에서도 동일하게 나타났다. 또한 비리온 단백질을 은염색 (silver stain)하여 Coomassie brilliant blue로 염색된 27개의 band외에 다수의 minor band를 관찰했다. 이들 단백질을 아미노산 분석한 결과는 다각체 단백질의 경우 glutamic acid, aspartic acid등의 양이 많은 반면 histidine, cystine등의 양은 비교적 적었으며, 뉴클레오캡시드는 주로 염기성인 arginine, lysine등이 많게 나타났다. 바이러스 DNA의 제한효소 분석에 의하면 그 genome은 약 122kb.였다. 한편 제한효소 분석 pattern중에 몇개의 submolar fragment가 관찰되었다. The structural proteins and DNA of Spodoptera litura nuclear polyhedrosis virus (SINPV) were characterised by SDS-PAGE, amino acid analysis and restriction endonucleases. The major band of MW.31 Kd and 7 minor polypeptides in the polyhedral protein were shown by SDS-PAGE. Viral proteins of SINPV were more detectable polypeptides by silver nitrate than 27 polypeptides by Coomassie brilliant blue. Among the above polypeptides, 12 polypeptides with small MW. were observed in the nucleocapsid protein. In amino acid analysis, glutamic acid and aspartic acid were abundant in the polyhedral protein while arginine and lysine in the nucleocapsid. The genome of SINPV was 122 Kb in size and several submolar fragments were observed by restriction endonucleases.
Kim Jinsoo,Hwang Seok Young,Kim Dongbum,Kim Minyoung,Baek Kyeongbin,Kang Mijeong,An Seungchan,Gong Junpyo,Park Sangkyu,Kandeel Mahmoud,Lee Younghee,Noh Minsoo,Kwon Hyung-Joo 한국응용약물학회 2022 Biomolecules & Therapeutics(구 응용약물학회지) Vol.30 No.5
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.
Hyun-Kyoung Lee,Byoung-Hee Lee,Seung-Hyeok Seok,Min-Won Baek,이희영,김동재,나이랑,Kyoung-Jin Noh,Sung-Hoon Park,Dutta Noton Kumar,Hiroaki Kariwa,Mina Nakauchi,Suk-Jin Heo,Jae-Hak Park 대한수의학회 2010 JOURNAL OF VETERINARY SCIENCE Vol.11 No.2
Severe acute respiratory syndrome (SARS) is a life-threatening disease for which accurate diagnosis is essential. Although many tools have been developed for the diagnosis of SARS, false-positive reactions in negative sera may occur because of cross-reactivity with other coronaviruses. We have raised polyclonal and monoclonal antibodies (Abs) using a recombinant form of the SARS virus nucleocapsid protein. Cross-reactivity of these anti-SARS Abs against human coronavirus (HCoV) 229E and HCoV OC43 were determined by Western blotting. The Abs produced reacted with recombinant SARS virus nucleocapsid protein, but not with HCoV 229E or HCoV OC43.
Jia-Qi Chu,Xu-Min Hu,김명철,박창식,전무형 한국미생물학회 2009 The journal of microbiology Vol.47 No.5
Three indirect enzyme-linked immunosorbent assays (iELISA) based on the North American like (NA-like), European like (EU-like) and co-expressed NA- and EU-like recombinant nucleocapsid proteins (N-protein) of porcine reproductive and respiratory syndrome virus (PRRSV) were validated for the detection of the antibodies in porcine sera. A total of 422 serum samples from unvaccinated pigs were tested. The cut-off value was optimized by a two-graph receiver operating characteristics analysis at a 95% confidence level. This assay was validated with Western blot analysis and IDEXX HerdChek™ ELISA. Cross-reactivity results showed that iELISA was PRRSV-specific. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were less than 10%. The results indicate that iELISA is simpler to produce and perform, time-saving and suitable for large scale surveys of PRRSV infection at low cost, and is potentially useful to evaluate the efficiency of various vaccines against PRRSV.
Kim, A.,Lee, J.Y.,Byun, S.J.,Kwon, M.H.,Kim, Y.S. Elsevier/North-Holland 2012 Antiviral research Vol.94 No.2
Influenza A virus infection is a great threat to avian species and humans. Targeting viral proteins by antibody has a limited success due to the antigen drift and shift. Here we present a novel antibody-based antiviral strategy of targeting viral genomic RNA (vRNA) for degradation rather than neutralizing viral proteins. Based on the template of a sequence-nonspecific nucleic acid-hydrolyzing, single domain antibody of the light chain variable domain, 3D8 VL, we generated a synthetic library on the yeast surface by randomizing putative nucleic acid interacting residues. To target nucleocapsid protein (NP)-encoding viral genomic RNA (NP-vRNA) of H9N2 influenza virus, the library was screened against a 18-nucleotide single stranded nucleic acid substrate, dubbed asNP<SUB>18</SUB>, the sequence of which is unique to the NP-vRNA. We isolated a 3D8 VL variant, NP25, that had ~15-fold higher affinity (~54nM) and ~3-fold greater selective hydrolyzing activity for the target substrate than for off targets. In contrast to 3D8 VL WT, asNP<SUB>18</SUB>-selective NP25 constitutively expressed in the cytosol of human lung carcinoma A549 cells does not exhibit any significant cytotoxicity and selectively degrades a reporter mRNA carrying the target asNP<SUB>18</SUB> sequence in the stable cell lines. NP25 more potently inhibits the replication of H9N2 influenza virus than 3D8 VL WT in the stable cell lines. NP25 more selectively reduces the amount of the targeted NP-vRNA than 3D8 VL WT from the early stage of virus infection in the stable cell lines, without noticeable harmful effects on the endogenous mRNA, suggesting that NP25 indeed more specifically recognizes to hydrolyze the target NP-vRNA of H9N2 virus than off-targets. Our results provide a new strategy of targeting viral genomic RNA for degradation by antibody for the prevention of influenza virus infection in humans and animals.
이창희 한국미생물·생명공학회 2009 한국미생물·생명공학회지 Vol.37 No.3
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 nonphosphorylated 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.
Xiaonan Fang,Lin-Bai Ye,Yijuan Zhang,Baozong Li,Shanshan Li,Lingbao Kong,Yuhua Wang,Hong Zheng,Wei Wang,Zhenghui Wu 한국미생물학회 2006 The journal of microbiology Vol.44 No.5
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.