• BMB Reports
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• 제44권6호
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• pp.393-398
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• 2011

Polydnaviruses are a group of double-stranded DNA viruses and are symbiotically associated with some ichneumonoid wasps. As proviruses, the replication of polydnaviruses occurs in the female reproductive organ at the pupal stage. This study analyzed the effects of two developmental hormones, juvenile hormone (JH) and ecdysteroid, on the viral replication of Cotesia plutellae bracovirus (CpBV). All 23 CpBV segments identified contained a conserved excision/rejoining site ('AGCTTT') from their proviral segments. Using quantitative real-time PCR based on this excision/rejoining site marker, initiation of CpBV replication was determined to have occurred on day 4 on the pupal stage. Pyriproxyfen, a JH agonist, significantly inhibited adult emergence of C. plutellae, whereas RH5992, an ecdysteroid agonist, had no inhibitory effect. Although RH5992 had no effect dose on adult development, it significantly accelerated viral replication. The results of immunoblotting assays against viral coat proteins support the effects of the hormone agonists on viral replication.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2003년도 International Meeting of the Microbiological Society of Korea
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• pp.83-86
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• 2003

Major advances in positive-sense RNA virus research have been facilitated by the development of reverse genetics systems. These systems consist of an infectious cDNA clone that encompasses the genome of the virus in question. This clone is then used as a template for the subsequent synthesis of infectious RNA for the generation of synthetic viruses. However, the construction of infectious cDNA for the Japanese encephalitis virus (JEV) has been repeatedly thwarted by the instability of its cDNA. As JEV is an important human pathogen that causes permanent neuropsychiatric sequelae and even fatal disease, a reliable reverse genetics system for this virus is highly desirable. The availability of this tool would greatly and the development of effective vaccines as well as facilitate studies into the basic biology of the virus, including the molecular mechanisms of viral replication, neurovirulence, and pathogenesis. We have successfully constructed a genetically stable infectious JEV cDNA containing full-length viral RNA genome. Synthetic RNA transcripts generated in vitro from the cDNA were highly infectious upon transfection into susceptible cells, and the cDNA remained stable after it had been propagated in E. coli for 180 generations. Using this infectious JEV cDNA, we have successfully expressed a variety of reporter genes from the full-length genomic and various subgenomic RNAs in vitro transcribed from functional JEV cDNAS. In summary, we have developed a reverse genetics system for JEV that will greatly facilitate the research on this virus in a variety of different fields. It will also be useful as a heterologous gene expression vector and aid the development of a vaccine against JEV.

• 한국자기공명학회논문지
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• 제20권3호
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• pp.66-70
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• 2016

The replication protein A (RPA), is a heterotrimer with 70, 32 and 14 kDa subunits and plays a crucial role in DNA replication, recombination, and repair. The largest subunit, RPA70, binds to single-stranded DNA (ssDNA) and mediates interactions with many cellular and viral proteins. In this study, we performed nuclear magnetic resonance experiments on the complex of the DNA binding domain A of human RPA70 (RPA70A) with ssDNA, d(CCCCC), at various temperatures, to understand the temperature dependency of ssDNA binding affinity of RPA70A. Essential residues for ssDNA binding were conserved while less essential parts were changed with the temperature. Our results provide valuable insights into the molecular mechanism of the ssDNA binding of human RPA.

• 대한수의학회지
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• 제57권1호
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• pp.37-42
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• 2017

Getah virus (GETV) infection causes sporadic outbreaks of mild febrile illness in horses and reproductive failure in pigs. In this study, we established a reverse transcription polymerase chain reaction (RT-PCR) method to detect GETV from suspected virus-infected samples. The reaction conditions were optimized and validated by using RNA extracted from GETV propagated in cell culture. A GETV-specific GED4 primer set was designed and used to amplify a 177 bp DNA fragment from a highly conserved region of the E1 glycoprotein gene in the GETV genome. RT-PCR performed with this primer set revealed high sensitivity and specificity. In the sensitivity test, the GED4 primer set detected GETV RNA at the level of $10^{2.0}\;TCID_{50}/mL$. In the specificity test, the GED4 primer set amplified only a single band of PCR product on the GETV RNA template, without non-specific amplification, and exhibited no cross-reactivity with other viral RNAs. These results suggest that this newly established RT-PCR method is useful for accurate identification of GETV infection in animals.

• 대한의생명과학회지
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• 제9권2호
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• pp.67-74
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• 2003

Viral and non-viral vectors have been used in the delivery of genetic materials into animal cells and tissues, with each approach having pros and cons. Non-viral vectors have many useful merits such as easy preparation, low immunity and size tolerance of a transgene when compared to those of viral vectors. Delivery specificity may be achieved by complex formation between receptor ligands and a non-viral vector. In the present study, non-viral vector systems are investigated in an effort to find a practical delivery means for gene therapy, Receptor-ligand interaction between transferrin-receptor and transferrin was utilized for efficient gene transfer into cancer cells. A plasmid vector, pcDNA3 (LacZ) was ligated with a small duplexed oligo fragment in which a Biotin- VN$^{TM}$ phosphoramidite was placed in the middle of the oligo. The plasmid vector labeled by biotin was then conjugated with biotin-labeled transferrin via streptavidin. This trimeric conjugates were delivered to a hepatoma cell line, HepG2. The delivery efficiency of the trimeric conjugate was 2-fold higher than that of cationic liposomes used for transfection of a plasmid vector. These results demonstrate that a plasmid vector can be efficiently transferred into cells by forming a trimeric complex of plasmid vector-linker-ligand.

• Journal of Microbiology and Biotechnology
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• 제32권12호
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• pp.1515-1526
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• 2022

Eukaryotic chromatin is highly organized in the 3D nuclear space and dynamically regulated in response to environmental stimuli. This genomic organization is arranged in a hierarchical fashion to support various cellular functions, including transcriptional regulation of gene expression. Like other host cellular mechanisms, viral pathogens utilize and modulate host chromatin architecture and its regulatory machinery to control features of their life cycle, such as lytic versus latent status. Combined with previous research focusing on individual loci, recent global genomic studies employing conformational assays coupled with high-throughput sequencing technology have informed models for host and, in some cases, viral 3D chromosomal structure re-organization during infection and the contribution of these alterations to virus-mediated diseases. Here, we review recent discoveries and progress in host and viral chromatin structural dynamics during infection, focusing on a subset of DNA (human herpesviruses and HPV) as well as RNA (HIV, influenza virus and SARS-CoV-2) viruses. An understanding of how host and viral genomic structure affect gene expression in both contexts and ultimately viral pathogenesis can facilitate the development of novel therapeutic strategies.

• 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.

• The Plant Pathology Journal
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• 제32권1호
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• pp.70-76
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• 2016

The infectious full-length cDNA clone of zucchini yellow mosaic virus (ZYMV) isolate PA (pZYMV-PA), which was isolated from pumpkin, was constructed by utilizing viral transcription and processing signals to produce infectious in vivo transcripts. Simple rub-inoculation of plasmid DNAs of pZYMV-PA was successful to cause infection of zucchini plants (Cucurbita pepo L.). We further engineered this infectious cDNA clone of ZYMV as a viral vector for systemic expression of heterologous proteins in cucurbits. We successfully expressed two reporter genes including gfp and bar in zucchini plants by simple rub-inoculation of plasmid DNAs of the ZYMV-based expression constructs. Our method of the ZYMV-based viral vector in association with the simple rub-inoculation provides an easy and rapid approach for introduction and evaluation of heterologous genes in cucurbits.

• 대한수의학회지
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• 제35권2호
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• pp.287-295
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• 1995

Molecular cloning and nucleotide sequencing were undertaken for the RNA genome of gp53 antigenic region in cytopathic Singer strain of bovine viral diarrhea virus. The cloned cDNA was 939 nucleotides in length having a base composition of 31.0% A, 19.6% C, 25.5% G and 24.0% T. The sequence was corresponded to approximately 77.8%(817 bases) of predicted gp53 region and 122 bases after 3'end of gp53 region in the Singer strain when compared with NADL strain of known sequence. A single open reading frame was found in the sequence of 2nd frame and was deduced as encoding 312 amino acids.

• Journal of Plant Biotechnology
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• 제7권3호
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• pp.143-148
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• 2005

The coat protein mediated resistance to potato virus Y is assessed here in transgenic potato plants (Solanum tuberosum L., cv Claustar). Therefore, the corresponding cDNA from tunisian isolate of the virus was cloned into Agrobacterium tumefaciens binary vector. The transgenic lines were subsequently analysed for the presence and expression of the transgene. The CP cDNA copy number was determined for kanamycin resistant plants. Three selected transgenic lines and their S1 progeny resulting from tuber germination showed a high protection level against the virus. These data appear to support the hypothesis that the virus resistance is mediated by the translated viral coat protein expressed in transgenic potato lines.