• BMB Reports
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• v.44 no.6
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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.

• Proceedings of the Microbiological Society of Korea Conference
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• 2003.05a
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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.

• Journal of the Korean Magnetic Resonance Society
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• v.20 no.3
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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.

• Korean Journal of Veterinary Research
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• v.57 no.1
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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.

• Biomedical Science Letters
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• v.9 no.2
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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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• v.32 no.12
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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.

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.86-95
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• 2002

As a result of genome projects, the research to elucidate the function of a protein of interest has recently been well-recognized. In order to facilitate functional genomics, a useful mammalian gene expression vector is required. Using an infectious CDNA clone of BVDV pNADLclns-, we have developed a mammalian gene expression vector. In this study, a replication-competent full-length infectious CDNA clone containing puremycin acetyltransferase (pac) gene (pNADLclns-/pac) was successfully generated. The viral RNA replication and viral protein NS3 synthesis were examined by detecting metabollically $^{32}P$-labelled genomic viral RNA and immunoblotting with a mouse anti-NS3 antibody. To generate viral replicon as an expression vector, we examine if the viral structural genes (C, E0, El, E2) are required for viral replication by deletion analysis. As a result, all of the structural proteins are dispensable for viral replication per se, but essential for infectious viral particle formation. Based on our deletion analysis, we have generated a replication-competent BVDV viral replicon (pNADLclns-/pac/${\Delta}S$), whose structural genes are all deleted. In addition to NADLclns- /pac/${\Delta}S$, NADLclns-/ luc/${\Delta}S$ viral replicon containing luciferase gene as a reporter was constructed and fecund to be replication-compotent in HeLa and BHK cells as well as MDBK cells. Therefore, BVDV viral replicon developed in our study will be a useful tool to express a protein of interest in various mammalian cells.

• The Plant Pathology Journal
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• v.32 no.1
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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.

• Korean Journal of Veterinary Research
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• v.35 no.2
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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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• v.7 no.3
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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.