• Journal of Microbiology and Biotechnology
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• 제24권12호
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• pp.1744-1754
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• 2014

The hepatitis C virus (HCV) RNA genome is replicated by an RNA replicase complex (RC) consisting of cellular proteins and viral nonstructural (NS) proteins, including NS5B, an RNA-dependent RNA polymerase (RdRp) and key enzyme for viral RNA genome replication. The HCV RC is known to be associated with an intracellular membrane structure, but the cellular components of the RC and their roles in the formation of the HCV RC have not been well characterized. In this study, we took a proteomic approach to identify stomatin, a member of the integral proteins of lipid rafts, as a cellular protein interacting with HCV NS5B. Co-immunoprecipitation and co-localization studies confirmed the interaction between stomatin and NS5B. We demonstrated that the subcellular fraction containing viral NS proteins and stomatin displays RdRp activity. Membrane flotation assays with the HCV genome replication-competent subcellular fraction revealed that the HCV RdRp and stomatin are associated with the lipid raft-like domain of membranous structures. Stomatin silencing by RNA interference led to the release of NS5B from the detergent-resistant membrane, thereby inhibiting HCV replication in both HCV subgenomic replicon-harboring cells and HCV-infected cells. Our results identify stomatin as a cellular protein that plays a role in the formation of an enzymatically active HCV RC on a detergent-resistant membrane structure.

• Journal of Microbiology and Biotechnology
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• 제26권4호
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• pp.637-647
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• 2016

Proliferating cell nuclear antigen (PCNA) is a critical eukaryotic replication accessory factor that supports DNA binding in DNA processing, such as DNA replication, repair, and recombination. PCNA consists of three toroidal-shaped monomers that encircle double-stranded DNA. The diverse functions of PCNA may be regulated by its interactions with partner proteins. Many of the PCNA partner proteins generally have a conserved PCNA-interacting peptide (PIP) motif, located at the N- or C- terminal region. The PIP motif forms a 3₁₀ helix that enters into the hydrophobic groove produced by an interdomain-connecting loop, a central loop, and a C-terminal tail in the PCNA. Post-translational modification of PCNA also plays a critical role in regulation of its function and binding partner proteins. Structural and biochemical studies of PCNA-protein will be useful in designing therapeutic agents, as well as estimating the outcome of anticancer drug development. This review summarizes the characterization of eukaryotic PCNA in relation to the protein structures, functions, and modifications, and interaction with proteins.

• Journal of Veterinary Science
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• 제21권5호
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• pp.72.1-72.10
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• 2020

Background: Fenbendazole, a dewormer drug, is used widely in the clinical treatment of parasite infections in animals. Recent studies have shown that fenbendazole has substantial effects on tumor growth, immune responses, and inflammatory responses, suggesting that fenbendazole is a pluripotent drug. Nevertheless, the antiviral effects have not been reported. Fenbendazole can disrupt microtubules, which are essential for multiple viruses infections, suggesting that fenbendazole might have antiviral effects. Objectives: This study examined whether fenbendazole could inhibit bovine herpesvirus 1 (BoHV-1) productive infection in cell cultures. Methods: The effects of fenbendazole on viral production, transcription of the immediate early (IE) genes, viron-associated protein expression, and the cellular signaling PLC-γ1/Akt pathway were assessed using distinct methods. Results: Fenbendazole could inhibit BoHV-1 productive infections significantly in MDBK cells in a dose-dependent manner. A time-of-addition assay indicated that fenbendazole affected both the early and late stages in the virus replication cycles. The transcription of IE genes, including BoHV-1 infected cell protein 0 (bICP0), bICP4, and bICP22, as well as the synthesis of viron-associated proteins, were disrupted differentially by the fenbendazole treatment. The treatment did not affect the cellular signaling pathway of PLC-γ1/Akt, a known cascade playing important roles in virus infection. Conclusions: Overall, fenbendazole has antiviral effects on BoHV-1 replication.

• 생약학회지
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• 제47권2호
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• pp.137-142
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• 2016

Enterovirus is a common cause of several severe diseases such as myocarditis, hand-foot-mouth disease, and meningitis in children and adult. There are many try to develop new antiviral drug for direct treatment in virus infection. However, synthetic chemical antiviral drug is not working. To overcome this limitation, we examined plant extracts. The antiviral effect of plant extracts was screened by HeLa cell survival assay in coxsackievirus B3 (CVB3) infection. We observed a strong antiviral effect of Poria cocos extract in a dose-dependent manner (1 mg/ml~0.01 mg/ml). P. cocos extract (1 mg/ml) treatment was dramatically decreased virus protease 2A induced eIF4G-I cleavage and virus capsid protein VP1 production. CVB3 positive and negative strand RNA amplification were significantly reduced in P. cocos extract treatment. P. cocos extract completely blocked early time activation of ERK and AKT activity in CVB3 infection. Taken together these data indicate that the treatment of P. cocos extract strongly inhibit CVB3 replication. Poria cocos extract may possible to developed as a therapeutic agent for enterovirus.

• Animal cells and systems
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• 제7권1호
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• pp.81-87
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• 2003

While screening proteins that interact with DnaA protein, the initiator protein for Escherichia coil chromosomal DNA replication, we found a 52-kD sized protein which bound to DnaA protein in a salt-dependent manner. This protein was identified as trigger factor, a ribosome-associated peptidyl-prolyl- cisltrans isomerase with chaperone activity. Trigger factor was overproduced and purified to near homogeneity, and its effect on the function of DnaA protein was examined, Enhanced binding of DnaA protein to DnaA box with no apparent supershift in the gel-shift experiments suggested that trigger factor, by virtue of its chaperone activity, exerts a change on DnaA protein thus increasing its binding affinity for DnaA box.

• 미생물학회지
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• 제38권2호
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• pp.86-95
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• 2002

최근 인간을 비롯한 다양한 생명체의 genome project연구결과 밝혀진 유전자들의 염기서열을 토대로, 생명체 구성성분의 실질적 인 역할을 하는 단배질의 기능을 밝히는 proteomics에 관한 연구의 필요성 이 대두되고 있다. 따라서, 이 연구는 post-genomics시대에 다양한 종류의 단백질 기능과 상호작용의 기초연구에 필수적 인 새로운 포유동물세포 유전자 발현벡터를 RNA 바이러스인 소설사성 바이러스(Bovine Viral Diarrhea Virus)의 infectious CDNA molecular clone을 이용하여 개발하였다. 먼저 BVDV의 infectious CDNA molecular clone (pNADLclns-)을 이용하여 puromycin 항생제에 저항성을 나타내는 puromycin acetyltransferase (pac) 유전자를 삽입하여 recombinant full-length infectious CDNA clone을 합성하였다. 합성된 recombinant CDNA clone을 주형으로 T7 RNA polymerase를 사용하여 in vitro transcribed full-length viral RNA를 합성하였다. 합성된 viral RNA의 자가복제 여부는 MDBK세포에 transfection시킨 후, $^{32}P$ 로 metabolically label함으로써 확인하였다. 또한, transfection된 세포에서의 바이러스 단백질 발현여부는 바이러스에 특이적으로 반응하는 anti-NS3 단클론항체를 사용하여 분석하였다. 또한, infectious CDNA clone을 응용하여 새로운 포유동물세포유전자 발현벡터의 개발을 위해서, 먼저 바이러스의 구조단백질이 바이러스의 자가복제에 필수적인 지를 평가하였다. 실험결과, 각각의 구조단백질 유전자를 deletion한 recombinant cDNA clone으로부터 합성된 viral RMA의 자가복제여부는pac유전자의 발현여부로 recombinant cDNA clone으로부터 합성된 recombinant viral RMA를 MDBK 세포에 transfection시킨 후, puromycin으로 selection함으로써 할 수 있었다. Deletion실험결과, 각각의 구조단백질 capsid및 E0, El, E2는 바이러스의 자가복제에 영향을 기치지 않음을 알 수 있었다. 이와 더불어, 바이러스의 모든 구조단백질을 함께deletion하였을 경우에도 자가복제에는 영향을 기치지 않는 것을 합성된 viral replicon을 이용한 실험에서 알 수 있었다. 이렇게 합성된 BVDV의 replicon을 사용하여 포유동물의 발현벡터로써 사용할수 있는 지의 여부를 분석하기 위해서 pac유전자 이외에 luciferase유전자를 사용하여 MDBK및 HeLa, BHK세포에서의 단백질 발현정도를 시간 별로 분석한 결과, BVDV의 replicon을 다양한 종류의 유전자 발현벡터로사용할 수 있음을 알 수 있었다. 그러므로, RNA바이러스의 하나인 BVDV의 viral replicon을 이용하여 다양한 종류의 포유동물 세포에 유전자 발현벡터로써 사용할 수 있음으로 post-genomics시대에 다양한 종류의 단백질 기능연구에 맡은 도움이 되리라 기대한다.

• Journal of Microbiology and Biotechnology
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• 제25권10호
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• pp.1768-1771
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• 2015

Human Fen1 protein (hFen1) plays an important role in Okazaki fragment processing by cleaving the flap structure at the junction between single-stranded (ss) DNA and doublestranded (ds) DNA, an intermediate formed during Okazaki fragment processing, resulting in ligatable nicked dsDNA. It was reported that hChlR1, a member of the cohesion establishment factor family, stimulates hFen1 nuclease activity regardless of its ATPase activity. In this study, we found that cohesion establishment factors cooperatively stimulate endonuclease activity of hFen1 in in vivo mimic condition, including replication protein-A-coated DNA and high salt. Our findings are helpful to explain how a DNA replication machinery larger than the cohesion complex goes through the cohesin ring structure on DNA during S phase in the cell cycle.

• 미생물학회지
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• 제36권2호
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• pp.103-108
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• 2000

한국에서 분리된 전염성 조혈괴저바이러스(infectious hematopoietic necrosis virus, IHNV)인 IHNV-PRT의 non-viron(NV)단백질을 암호화하고 있는 cDNA를 클로닝하여 이들의 염기서열을 분석하였다. NV는 336bpzmrl의 open reading frame을 포함하였으며 이로부터 111개의 아미노산 서열을 외국에서 분리된 IHNV들과 비교 분석한 결과 90-95%의 상동성을 보였다. 이러한 사실은 INHV의 NV단백질 유전자들은 IHNV의 strain에 관계없이 매우 보존되어 있음을 나타내준다. Northern blotting을 사용하여 NV의 발현을 측정한 결과 감염 후 20 시간분터 발현이 증가함을 확인 힐수 있었다. NV가 바이러스의 증식에 필요한지의 여부를 확인하기 위하여 바이러스 유전자의 antisense DNA를 사용하여 바이러스 증식 억제에 관한 실험을 수행하였다. Glycoprotein (G)의 antisense DNA를 처리한 경우 바이러스의 증식이 거의 억제된 반면 NV에 대한 antisense DNA를 처리한 경우 바이러스 증식에 거의 변화가 없었다. 이로부터 배양중인 세포가 있어서 NV는 증식에 필수적이지 않은 것으로 판단된다.

• 한국자기공명학회논문지
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• 제20권1호
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• pp.22-26
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• 2016

FANCJ is a DNA helicase which contributes genome stability by resolving G-quadruplex DNA from 5' to 3' direction. In addition to main ATPase helicase core, FANCJ has the protein binding region at its C-terminal part. BRCA1 and BLM are the binding partner of FANCJ and these protein-protein interactions contribute genomic stability and the proper response to replication stress. As the first attempt for studying FANCJ-BLM interaction, we prepared BLM binding region of FANCJ and characterized with CD and NMR spectroscopy. FANCJ (881-941) with N-ter 6xHis was purified as the oligomer. Secondary structure prediction based on CD data revealed that FANCJ (881-941) composed with ${\beta}$ sheet, turn and coils.$^1H-^{15}N$ HSQC spectra showed nonhomogeneous peak intensities with less number of peaks comparing than the number of amino acids in the construct. It indicated that optimization should be necessary for detailed further structural studies.

• Journal of Microbiology
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• 제41권4호
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• pp.300-305
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• 2003

Intracellular expression of RNA decoys, such as TAR or RRE decoy, has been previously shown to protect immune cells from human immunodeficiency virus type 1 (HIV-1) replication by inhibiting the binding of the HIV-1 regulatory protein to the authentic HIV RNA sequence. However, HIV-1 challenge experiments of primary human T cells, which express the RNA decoy, demonstrated that the cells were only transiently protected, and hence, more improved protocols for HIV-1 inhibition with the RNA decoys need to be developed. In this report, in order to develop a more effective RNA decoy, we analyzed and compared the ability of a series of RNA decoy derivatives in inhibiting HIV-1 replication in CEM cells. Using an improved tRNA cassette to express high levels of RNA decoy transcripts in cells, we found that co-expression of both TAR and RRE decoys, in the form of an aligned sequence in a single transcription cassette, much more potently blocked cells from HIV-1 than the expression of only one kind of RNA decoy. This observation will have an important implication for experiments involving optimization of clinical applications in RNA decoy-based gene therapy against HIV-1.