• Bulletin of the Korean Chemical Society
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• v.20 no.3
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• pp.301-306
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• 1999

Hepatitis C virus (HCV) has been known to be an enveloped virus with a positive strand RNA genome and the major agent of the vast majority of transfusion associated cases of hepatitis. For viral replication, HCV structural proteins are first processed by host cell signal peptidases and NS2/NS3 site of the nonstructural protein is cleaved by a zinc-dependent protease NS2 with N-terminal NS3. The four remaining junctions are cleaved by a separate NS3 protease. The solution conformations of NS4B/5A, NS5A/5B substrates and NS5A/5B inhibitor have been determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. NMR data suggested that the both NS5A/5B substrate and inhibitor appeared to have a folded tum-like conformation not only between P1 and P6 position but also C-terminal region, whereas the NS4B/5A substrate exhibited mostly extended conformation. In addition, we have found that the conformation of the NS5A/5B inhibitor slightly differs from that of NS5A/5B substrate peptide, suggesting different binding mode for NS3 protease. These findings will be of importance for designing efficient inhibitor to suppress HCV processing.

• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1651-1658
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• 2020

Since Zika virus (ZIKV) was first detected in Uganda in 1947, serious outbreaks have occurred globally in Yap Island, French Polynesia and Brazil. Even though the number of infections and spread of ZIKV have risen sharply, the pathogenesis and replication mechanisms of ZIKV have not been well studied. ZIKV, a recently highlighted Flavivirus, is a mosquito-borne emerging virus causing microcephaly and the Guillain-Barre syndrome in fetuses and adults, respectively. ZIKV polyprotein consists of three structural proteins named C, prM and E and seven nonstructural proteins named NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 in an 11-kb single-stranded positive sense RNA genome. The function of individual ZIKV genes on the host innate immune response has barely been studied. In this study, we investigated the modulations of the NF-κB promoter activity induced by the MDA5/RIG-I signaling pathway. According to our results, two nonstructural proteins, NS2A and NS4A, dramatically suppressed the NF-κB promoter activity by inhibiting signaling factors involved in the MDA5/RIG-I signaling pathway. Interestingly, NS2A suppressed all components of MDA5/RIG-I signaling pathway, but NS4A inhibited most signaling molecules, except IKKε and IRF3-5D. In addition, both NS2A and NS4A downregulated MDA5-induced NF-κB promoter activity in a dosedependent manner. Taken together, our results suggest that NS2A and NS4A signifcantly antagonize MDA5/RIG-I-mediated NF-κB production, and these proteins seem to be controlled by different mechanisms. This study could help understand the mechanisms of how ZIKV controls innate immune responses and may also assist in the development of ZIKV-specific therapeutics.

• Molecules and Cells
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• v.22 no.1
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• pp.13-20
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• 2006

Hepatitis C virus (HCV) non-structural protein 5A protein (NS5A), which consists of three functional domains, is involved in regulating viral replication, interferon resistance, and apoptosis. Recently, the three-dimensional structure of the domain 1 was determined. However, currently the molecular basis for the domains 2 and 3 of HCV NS5A is yet to be defined. Toward this end, we expressed, purified the domain 2 of the NS5A (NS5A-D2), and then performed biochemical and structural studies. The purified domain 2 was active and was able to bind NS5B and PKR, biological partners of NS5A. The results from gel filtration, CD analysis, 1D $^1H$ NMR and 2D $^1H-^{15}N$ heteronuclear single quantum correlation (HSQC) spectroscopy indicate that the domain 2 of NS5A appears to be flexible and disordered.

• Journal of Sensor Science and Technology
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• v.12 no.1
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• pp.1-9
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• 2003

CsI single crystals doped with lithium, potassium or rubidium were grown by using Czochralski method at Ar gas atmosphere. The energy resolutions of CsI(Li:0.2 mole%), CsI(K:0.5 mole%) and CsI(Rb:1.5 mole%) scintillators were 14.5%, 15.9% and 17.0% for $^{137}Cs$(0.662 MeV), respectively. The energy calibration curves of CsI(Li), CsI(K) and CsI(Rb) scintillators were linear for $\gamma$-ray energy. The time resolutions of CsI(Li:0.2 mole%), CsI(K:0.5 mole%) and CsI(Rb:1.5 mole%) scintillators measured by CFT(constant-fraction timing method) were 9.0 ns, 14.7 ns and 9.7 ns, respectively. The fluorescence decay times of CsI(Li:0.2 mole%) scintillator had a fast component and slow one of ${\tau}_1=41.2\;ns$ and ${\tau}_2=483\;ns$, respectively. The fluorescence decay times of CsI(K:0.5 mole%) scintillator were ${\tau}_1=47.2\;ns$ and ${\tau}_2=417\;ns$. And the fluorescence decay times of CsI(Rb:1.5 mole%) scintillator were ${\tau}_1=41.3\;ns$ and ${\tau}_2=553\;ns$. The phosphorescence decay times of CsI(Li:0.2 mole%), CsI(K:0.5 mole%) and CsI(Rb:1.5 mole%) scintillators were 0.51 s, 0.57 s and 0.56 s, respectively.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.115-128
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• 1997

Three cDNA fragments located within NS5 region of HCV were synthesized by RT using viral RNA extracted from blood sample of Korean patient as a template. The cDNAs were amplified by PCR, cloned into the T-vector, and the nucleotide sequences were determined. Comparative analysis of the nucleotide and amino acid sequence of NS5 cDNAs showed that it is closely related with HCV type 1b. The cloned NS5 cDNA showed 91-94% homology at the nucleotide sequence level and 96-98% homology at the amino acid sequence level with several strains of the HCV type 1b. The NS5 cDNAs were subcloned into E. coli expression vectors to construct pRSETA5-1, pTHAN5-1, pRSETC5-2, pRSETBB1, pRESTCB1 and pRSETB-H3. Expression of the NS5 proteins was achieved by inducing the promoter with isopropyl-thio-${\beta}$-D-galactoside (IPTG) and confirmed by SDS-polyacrylamide gel electrophoresis. The NS5 proteins were immunoreactive against sera from Korean hepatitis C patients in Western blot analysis. Among the recombinant NS5 proteins, pRSETAS-1 plasmid derived protein, coded from aa2022 to aa2521 of HCV polyprotein, showed the strongest immunoreactivity against sera from Korean hepatitis C patients in immunoblot analysis. These results suggest that NS5 proteins would be useful as an antigen for detection of antibody against HCV in the blood samples.

• BMB Reports
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• v.37 no.6
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• pp.741-748
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• 2004

The hepatitis C virus is associated with the development of liver cirrhosis and hepatocellular carcinomas. Among the 10 polyproteins produced by the virus, no function has been clearly assigned to the non-structural 5A (NS5A) protein. This study was designed to identify the hepatocellular proteins that interact with NS5A of the HCV. Yeast two-hybrid experiments were performed with a human liver cDNA prey-library, using five different NS5A derivatives as baits, the full-length NS5A (NS5A-F, amino acid (aa) 1~447) and its four different derivatives, denoted as NS5A-A (aa 1~150), -B (aa 1~300), -C (aa 300~447) and D (aa 150~447). NS5A-F, NS5A-B and NS5A-C gave two, two and 10 candidate clones, respectively, including an AHNAK-related protein, the secreted frizzled-related protein 4 (SFRP4), the N-myc downstream regulated gene 1 (NDRG1), the cellular retinoic acid binding protein 1 (CRABP-1), ferritin heavy chain (FTH1), translokin, tumor-associated calcium signal transducer 2 (TACSTD2), phosphatidylinositol 4-kinase (PI4K) and $centaurin{\delta}$ 2 ($CENT{\delta}2$). However, NS5A-A produced no candidates and NS5A-D was not suitable as bait due to transcriptional activity. Based on an in vitro binding assay, CRABP-1, PI4K, $CENT{\delta}2$ and two unknown fusion proteins with maltose binding protein (MBP), were confirmed to interact with the glutathione S-transferase (GST)/NS5A fusion protein. Furthermore, the interactions of CRABP-1, PI4K and $CENT{\delta}2$ were not related to the PXXP motif (class II), as judged by a domain analysis. While their biological relevance is under investigation, the results contribute to a better understanding of the possible role of NS5A in hepatocellular signaling pathways.

• Korean Journal of Microbiology
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• v.47 no.3
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• pp.188-193
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• 2011

As a specific and effective therapeutic genetic material against hepatitis C virus (HCV) multiplication, HCV internal ribosome entry site (IRES)-targeting hammerhead ribozyme which activity is allosterically regulated by HCV regulatory protein, NS5B RNA replicase, was constructed. The allosteric ribozyme was composed of sequence of RNA aptamer to HCV NS5B, communication module sequence which can transfer structural transition for inducing ribozyme activity upon binding NS5B to the aptamer, and sequence of ribozyme targeting +382 nucleotide of HCV IRES. With real-time PCR analysis, the ribozyme was found to efficiently inhibit HCV replicon replication in cells. Of note, the allosteric ribozyme was shown to inhibit HCV replicon replication more efficiently than either HCV genome-targeting ribozyme or NS5B aptamer only. This allosteric ribozyme can be used as a lead genetic agent for the specific and effective suppression of HCV replication.

• Molecules and Cells
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• v.40 no.3
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• pp.202-210
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• 2017

The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus (HCV) RNA genome is a multifunctional phosphoprotein. To analyse the influence of NS5A on apoptosis, we established an Hep-NS5A cell line (HepG2 cells that stably express NS5A) and induced apoptosis using tumour necrosis factor $(TNF)-{\alpha}$. We utilised the MTT assay to detect cell viability, real-time quantitative polymerase chain reaction and Western blot to analyse gene and protein expression, and a luciferase reporter gene experiment to investigate the targeted regulatory relationship. Chromatin immunoprecipitation was used to identify the combination of $NF-{\kappa}B$ and miR-503. We found that overexpression of NS5A inhibited $TNF-{\alpha}$-induced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the $TNF-{\alpha}$ induced Hep-mock cells was significantly less than the viability of the $TNF-{\alpha}$ induced Hep-NS5A cells, which demonstrates that NS5A inhibited $TNF-{\alpha}$-induced HepG2 cell apoptosis. Under $TNF-{\alpha}$ treatment, miR-503 expression was decreased and cell viability and B-cell lymphoma 2 (bcl-2) expression were increased in the Hep-NS5A cells. Moreover, the luciferase reporter gene experiment verified that bcl-2 was a direct target of miR-503, NS5A inhibited $TNF{\alpha}$-induced $NF-{\kappa}B$ activation and $NF-{\kappa}B$ regulated miR-503 transcription by combining with the miR-503 promoter. After the Hep-NS5A cells were transfected with miR-503 mimics, the data indicated that the mimics could reverse $TNF-{\alpha}$-induced cell apoptosis and blc-2 expression. Collectively, our findings suggest a possible molecular mechanism that may contribute to HCV treatment in which NS5A inhibits $NF-{\kappa}B$ activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis.

• The Korean Journal of Mycology
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• v.44 no.3
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• pp.138-144
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• 2016

Twelve unrecorded yeasts, Pseudozyma prolifica HL9-1, Trichosporon coremiiforme NS19-2, Candida cretensis SA4-1, Cryptococcus diffluens TJ4-3, Cryptococcus pinus YB17-2, Candida vartiovaarae DD2-5, Pichia galeiformis DM3-5, Candida pseudolambica JW2-3, Trichosporon xylopini NS5-1, Trichosporon moniliiforme NS5-7, Tetrapisispora iriomotensis NS14-2, and Tetrapisispora nanseiensis SA17-1, were screened among 97 yeasts from soils of Chungcheongnam-do and Daejeon metropolitan city, Korea. These yeasts were oval or ellipsoidal and had a budding system for vegetative reproduction. They grew well in yeast extract-peptone-dextrose (YPD) medium and, in particular, Tetrapisispora iriomotensis NS14-2 and Candida cretensis SA4-1 grew well in 10% NaCl-containing YPD broth. Nine strains, including Trichosporon coremiiforme NS19-2, assimilated xylose and four yeast strains, such as Candida vartiovaarae DD2-5, also assimilated lactose. Physiological functionalities of cell-free extracts and supernatants from two halophilic unrecorded yeasts, Candida cretensis SA4-1 and Tetrapisispora iriomotensis NS14-2, were investigated. Cell-free extracts from Candida cretensis SA4-1 and Tetrapisispora iriomotensis NS14-2 exhibited 71.3% and 68.4% antihypertensive angiotensin I-converting enzyme inhibitory activity.

• Molecules and Cells
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• v.21 no.3
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• pp.330-336
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• 2006

Since its identification in 1989, hepatitis C virus has been the subject of extensive research. The biology of the virus and the development of antiviral drugs are closely related. The RNA polymerase activity of nonstructural protein 5B was first demonstrated in 1996. NS5B is believed to localize to the perinuclear region, forming a replicase complex with other viral proteins. It has a typical polymerase structure with thumb, palm, and finger domains encircling the active site. A de novo replication initiation mechanism has been suggested. To date, many small molecule inhibitors are known including nucleoside analogues, non-nucleoside analogues, and pyrophosphate mimics. NS5B interacts with other viral proteins such as core, NS3, 4A, 4B, and 5A. The helicase activity of NS3 seems necessary for RNA strand unwinding during replication, with other nonstructural proteins performing modulatory roles. Cellular proteins interacting with NS5B include VAMP-associated proteins, heIF4AII, hPLIC1, nucleolin, PRK2, ${\alpha}$-actinin, and p68 helicase. The interactions of NS5B with these proteins might play roles in cellular trafficking, signal transduction, and RNA polymerization, as well as the regulation of replication/translation processes.