• IMMUNE NETWORK
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• 제1권1호
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• pp.77-86
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• 2001

Background: Hepatitis C virus(HCV), a family of Flaviviridae, has a host cell-derived envelope containing a positive-stranded RNA genome, and has been known as the maj or etiological agent for chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. There remains a need to dissect a molecular mechanism of pathogenesis for the development of therapeutic and effective preventive measure for HCV. Identification of cellular receptor is of central importance not only to understand the viral pathogenesis, but also to exploit strategies for prevention of HCV. This study was aimed at identifying peptide mimotopes inhibiting the binding of E2 protein of HCV to MOLT-4 cell. Methods: In this study, phage peptide library displaying a random peptides consisting of 7 or 12 random peptides was employed in order to pan against E2 protein. Free HCV particles were separated from the immune complex forms by immunoprecipitation using anti-human IgG antibody, and used for HCV-capture ELISA. To identify the peptides inhibiting E2-binding to MOLT-4 cells, E2 protein was subj ect to bind to MOLT-4 cells under the competition with phage peptides. Results: Several phage peptides were selected for their specific binding to E2 protein, which showed the conserved sequence of SHFWRAP from 3 different peptide sequences. They were also able to recognize the HCV particles in the sera of HCV patients captured by monoclonal antibody against E2 protein. Two of them, showing peptide sequence of HLGPWMSHWFQR and WAPPLERSSLFY respectively, were revealed to inhibit the binding of E2 protein to MOLT-4 cell efficiently in dose dependent mode. However, few membrane-associated receptor candidates were seen using Fasta3 programe for homology search with these peptides. Conclusion: Phage peptides containing HLGPWMSHWFQR and WAPPLERSSLFY respectively, showed the inhibition of E2-binding to MOLT-4 cells. However, they did not reveal any homologues to cellular receptors from GenBank database. In further study, cellular receptor could be identified through the screening of cDNA library from MOLT-4 or hepatocytes using antibodies against these peptide mimotopes.

• 미생물학회지
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• 제45권3호
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• pp.246-250
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• 2009

레트로바이러스는 바이러스 외막과 숙주세포막의 융합에 의해 세포내로 들어간다. Moloney 마우스레트로바이러스(murine leukemia virus)의 외막 단백질은 표면단백질(SU)과 막단백질(TM)을 포함하는 85 kDa의 전구체로 합성되는데 바이러스의 성숙과정에 막단백질의 카르복시 말단 16개의 아미노산(R-peptide)이 바이러스의 프로티아제에 의해 절단된다. R 펩타이드가 절단된 막단백질은 Moloney 마우스레트로바이러스에 대한 수용체를 가진 NIH3T3 세포주에서 합포체(syncytium)를 형성한다. R 펩타이드가 절단된 막단백질의 합포체 형성 기작 연구를 위해 R 펩타이드가 절단되어 있으며 표면단백질의 PRR (proline rich region) 부위가 EGFP로 삽입되어진 Moloney full length molecular clone을 만들었다. 이 clone은 NIH3T3 세포에서 합포체를 형성하였으며 형광이 세포질과 세포막에서 관찰되었으나 핵은 염색이 되지 않고 검게 보여 신속 정확하게 합포체 관찰이 가능하였다. 흥미롭게도 절단된 막단백질을 가진 비리온이 NIH3T3 세포에서 광학현미경으로 관찰하였을때는 합포체를 형성하였으나 형광현미경에서는 형광이 관찰되지 않아서 비리온이 세포감염 없이 바이러스-세포 융합 방식으로 합포체를 형성한 것으로 생각되었다. 본 연구에서는 형광의 발현 여부로 합포체 형성을 신속 정확하게 관찰할 수 있는 방법을 개발하였으며 R 펩타이드가 절단된 비리온이 세포 감염 없이 세포와 세포 사이의 융합을 매개할 수 있음을 밝혔다.

• Journal of Microbiology and Biotechnology
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• 제27권6호
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• pp.1098-1105
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• 2017

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones ($Mut^s$) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

• Journal of Microbiology and Biotechnology
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• 제18권12호
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• pp.1884-1889
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• 2008

The ${\beta}$-lactamase inhibitory protein, BLIP-II, found in the culture supernatant of Streptomyces exfoliatus SMF19, shows no discernible sequence identity with other ${\beta}$-lactamase inhibitory proteins identified in Streptomyces spp. A null mutant of the gene encoding BLIP-II (bliB::$hyg^r$) showed a bald appearance on solid media. Although BLIP-II was initially isolated from the supernatant of submerged cultures, sites of BLIP-II accumulation were seen in the cell envelope. Mutation of bliB was also associated with changes in the formation of septa and condensation of the chromosomal DNA associated with sporulation. The bliB mutant exhibited infrequent septa, showing dispersed chromosomal DNA throughout the mycelium, whereas the condensed chromosomes of the wild-type were separated by regularly spaced septa giving the appearance of a string of beads. Therefore, on the basis of these results, it is suggested that BLIP-II is a regulator of morphological differentiation in S. exfoliatus SMF19.

• Molecules and Cells
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• 제28권5호
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• pp.479-487
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• 2009

Glutathione reductase (GR) is an enzyme that recycles a key cellular antioxidant molecule glutathione (GSH) from its oxidized form (GSSG) thus maintaining cellular redox homeostasis. A recombinant plasmid to overexpress a GR of Brassica rapa subsp. pekinensis (BrGR) in E. coli BL21 (DE3) was constructed using an expression vector pKM260. Expression of the introduced gene was confirmed by semi-quantitative RT-PCR, immunoblotting and enzyme assays. Purification of the BrGR protein was performed by IMAC method and indicated that the BrGR was a dimmer. The BrGR required NADPH as a cofactor and specific activity was approximately 458 U. The BrGR-expressing E. coli cells showed increased GR activity and tolerance to $H_2O_2$, menadione, and heavy metal ($CdCl_2$, $ZnCl_2$ and $AlCl_2$)-mediated growth inhibition. The ectopic expression of BrGR provoked the co-regulation of a variety of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. Consequently, the transformed cells showed decreased hydroperoxide levels when exposed to stressful conditions. A proteomic analysis demonstrated the higher level of induction of proteins involved in glycolysis, detoxification/oxidative stress response, protein folding, transport/binding proteins, cell envelope/porins, and protein translation and modification when exposed to $H_2O_2$ stress. Taken together, these results indicate that the plant GR protein is functional in a cooperative way in the E. coli system to protect cells against oxidative stress.

• 한국가축번식학회지
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• 제27권1호
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• pp.15-23
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• 2003

본 연구는 VSV-G glycoprotein을 envelope으로 하는 pseudotyped retrovirus vector system을 이용하여 쥐의 유방상피세포인 HC11에서 human Lactadherin 유전자의 발현을 확인하고자 하였다. 실험에 사용한 vector는 개체내에서의 외래 유전자의 지속적인 발현에 의한 생리적인 부작용을 최소화하기 위한 구조로, 조직특이적이며 lactogenic hormone에 의해 유도적인 활성을 가지는 것으로 알려진 WAP promoter의 통제하에 도입하고자 하는 외래 유전자를 위치하도록 하였다. WAP promoter의 대조군으로 지속적인 활성을 나타내는 $\beta$-actin promoter를 사용하였으며, 이 각각의 promoter와 marker gene으로 E. coli LacZ gene을 재조합한 후 retrovirus vector system을 이용하여 HCll에 도입하였다. 세포의 genome 내로의 유전자의 전이는 PCR을 통해 확인하였고, RT-PCR의 수행으로 유전자의 발현을 확인하였다. Lactadherin 유전자를 이용한 실험도 동일한 과정으로 수행하였으며, RT-PCR의 결과에서 HCll 세포에서 Lactadherin 유전자의 발현이 insulin을 단독으로 처리한 군에 비해 insulin, hydrocortisone, prolactin을 동시에 처리한 군에서 우월하게 나타나는 것으로 확인되었다. 그러나 insulin 단독 처리군에서 유전자의 발현이 약하게 나타나는 것으로 관찰되어 WAP promoter의 leakiness에 대한 재고의 필요성이 요구되었다.

• 대한바이러스학회지
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• 제28권4호
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• pp.383-391
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• 1998

A murine monoclonal antibody (mAb) specific for the envelope glycoprotein gp120 of human immunodeficiency virus type-I (HIV -1) was chemically coupled to pokeweed antiviral protein (PAP) from Phytolacca americana. The immunotoxin was purified by FPLC using S200 colum. The purified immunotoxin efficiently bound to HIV-infected T cells as evidenced by fluorescenceactivated cell sorter analysis. The immunotoxin selectively killed human T lymphoid lines infected with $HIV-1_{IIIB}$ at less than 250 pM of the immunotoxin cells, while PAP or mAb alone did not have any significant effect on infected cells. The uninfected control T cell lines were not affected. Human cells infected with HIV-2 or other HIV-1 strains were not killed, suggesting that the killing depends completely on the antibody used for coupling. These in vitro results suggest that the PAP-mAb conjugate may be used to selectively remove cells expressing viral antigens from individuals infected with HIV.

• The Plant Pathology Journal
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• 제38권4호
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• pp.410-416
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• 2022

Erwinia amylovora, the causal agent of fire-blight disease in apple and pear trees, was first isolated in South Korea in 2015. Although numerous studies, including omics analyses, have been conducted on other strains of E. amylovora, studies on South Korean isolates remain limited. In this study, we conducted a comparative proteomic analysis of the strain TS3128, cultured in three media representing different growth conditions. Proteins related to virulence, type III secretion system, and amylovoran production, were more abundant under minimal conditions than in rich conditions. Additionally, various proteins associated with energy production, carbohydrate metabolism, cell wall/membrane/envelope biogenesis, and ion uptake were identified under minimal conditions. The strain TS3128 expresses these proteins to survive in harsh environments. These findings contribute to understanding the cellular mechanisms driving its adaptations to different environmental conditions and provide proteome profiles as reference for future studies on the virulence and adaptation mechanisms of South Korean strains.

• BMB Reports
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• 제53권3호
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• pp.166-171
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• 2020

A chemical library comprising 2,354 drug-like compounds was screened using a transcription and replication-competent viruslike particle (trVLP) system implementing the whole Ebola virus (EBOV) life cycle. Dose-dependent inhibition of Ebola trVLP replication was induced by 15 hit compounds, which primarily target different types of G protein-coupled receptors (GPCRs). Based on the chemical structure, the compounds were divided into three groups, diphenylmethane derivatives, promazine derivatives and chemicals with no conserved skeletons. The third group included sertindole, raloxifene, and ibutamoren showing prominent antiviral effects in cells. They downregulated the expression of viral proteins, including the VP40 matrix protein and the envelope glycoprotein. They also reduced the amount of EBOV-derived tetracistronic minigenome RNA incorporated into progeny trVLPs in the culture supernatant. Particularly, ibutamoren, which is a known agonist of growth hormone secretagogue receptor (GHSR), showed the most promising antiviral activity with a 50% effective concentration of 0.2 μM, a 50% cytotoxic concentration of 42.4 μM, and a selectivity index of 222.8. Here, we suggest a strategy for development of anti-EBOV therapeutics by adopting GHSR agonists as hit compounds.

• Asian-Australasian Journal of Animal Sciences
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• 제14권2호
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• pp.163-169
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• 2001

Despite the high potency of the retrovirus vector system in gene transfer, one of the main drawbacks of has been difficulty in preparing highly concentrated virus stock. Numerous efforts to boost the virus titer have ended in unsatisfactory results mainly due to fragile property of retrovirus envelope protein. In this study, to overcome this problem, we constructed our own retrovirus vector system producing vector viruses encapsulated with VSV-G (vesicular stomatitis virus G glycoprotein). Concentration process of the virus stock by ultracentrifuge did not sacrifice the virus infectivity, resulting in more than 108 to 109 CFU (colony forming unit) per ml on most of the target cell lines tested. Application of this high-titer retrovirus vector system was tested on chicken embryos. Injection of virus stock beneath the blastoderms of pre-incubated fertilized eggs resulted in chick embryos expressing E. coli LacZ gene with 100% efficiency. Therefore, our results suggest that it is possible to transfer the foreign gene into chicken embryo using our high-titer retrovirus vector.