• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.149.1-149
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• 2003

A lily strain of Cucumber mosaic virus (LK-CMV) was not able to systemically infect zucchini squash (Cucurbita pepo), while Fny strain of CMV (Fny-CMV) caused systemic mosaic and stunting symptom at 4 days post-inoculation on the same host species. The pathogenicity of LK-CMV in zucchini squash was investigated by reassortments of genomic RNAs of LK-CMV and Fny-CMV for infection, as well as by pseudorecombinants generated from biologically active transcripts of CDNA clones of LK-CMV and Fny-CMV, respectively. The assessments of pathogenicity for LK-CMV indicated that RNA2 of LK-CMV was responsible for systemic infection in zucchini squash. In the protoplast of zucchini squash, the RNA accumulations of all constructed pseudorecombinants were indistinguishable and LK-CMV replication was slightly lower than that of Fny-CMV, suggesting that the inability of LK-CMV to infect squash plants was responsible for the poor efficiency of virus movement, rather than the reduction of replication function.

• 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.

• Molecules and Cells
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• v.21 no.1
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• pp.112-120
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• 2006

It has been suggested that defective interfering (DI) RNA contributes to the persistence of Japanese encephalitis virus (JEV). In this study, we characterized molecular and biological aspects of the DI RNA and its relation to viral persistence. We identified a homologous DI virus intimately associated with JEV persistence in Vero cells. The production of DI RNA during undiluted serial passages of JEV coincided with the appearance of cells refractory to acute infection with JEV. We also established a Vero cell clone with a persistent JEV infection in which the DI RNA coreplicated efficiently at the expense of helper virus. The infectious virus yield of the clone fluctuated during its growth depending upon the amount of DI RNA accumulated in the previous replication cycle. Identification of the corresponding negative-sense RNA of the DI RNA indicated that the DI RNA functioned as a replication unit. Most of the DI RNA molecules retained their open reading frames despite a large deletion, encompassing most of the prM, the entire E, and the 5' half of the NS1 gene. Taken together, these observations suggest that the generation of homologous DI RNA during successive JEV acute infections in Vero cells probably participates actively in persistent JEV infection.

• Research in Plant Disease
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• v.27 no.4
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• pp.137-148
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• 2021

Plant RNA viruses are one of the most destructive pathogens that cause a significant loss in crop production worldwide. They have evolved with high genetic diversity and adaptability due to the short replication cycle and high mutation rate during genome replication, which are characteristics of RNA viruses. Plant RNA viruses exist as quasispecies with high genetic diversity; thereby, a rapid population transition with new fitness can occur due to selective pressure resulting from environmental changes. Plant resistance can act as selective pressure and affect the fitness of the virus, which may lead to the emergence of resistance-breaking variants. In this paper, we introduced the evolutionary perspectives of plant RNA viruses and the driving forces in their evolution. Based on this, we discussed the mechanism of the emergence of variant viruses that overcome plant resistance. In addition, strategies for deploying plant resistance to viral diseases and improving resistance durability were discussed.

• The Plant Pathology Journal
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• v.36 no.3
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• pp.280-288
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• 2020

RNA interference (RNAi) has attracted attention as a promising approach to control plant viruses in their insect vectors. In the present study, to suppress replication of the rice stripe virus (RSV) in its vector, Laodelphax striatellus, using RNAi, dsRNAs against L. striatellus genes that are strongly upregulated upon RSV infection were delivered through a rice leaf-mediated method. RNAi-based silencing of peroxiredoxin, cathepsin B, and cytochrome P450 resulted in significant down regulation of the NS3 gene of RSV, achieving a transcriptional reduction greater than 73.6% at a concentration of 100 ng/μl and, possibly compromising viral replication. L. striatellus genes might play crucial roles in the transmission of RSV; transcriptional silencing of these genes could suppress viral replication in L. striatellus. These results suggest effective RNAi-based approaches for controlling RSV and provide insight into RSV-L. striatellus interactions.

• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.31-31
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• 1996

The double-stranded panhandle structure of the influenza virus RNA is important for the replication, transcription and packaging into the virion of the vRNA. The solution structure of a 34-nucleotide-long RNA which contains the conserved panhandle sequences has been investigated by one- and two-dimensional NMR spectroscopies. (omitted)

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.96-102
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• 2002

The S-phase checkpoint mechanisms response to DNA damage or inhibition of DNA replication for maintenance of genetic stability in eukaryotic cells. These roles include cell cycle control arrest at S-phase and Iranscriptional induction of repair genes. To characterize the defects of dpbll mutant for both these responses, we examined the over-expression effect of DPBll gene, the sensitivity to HU, MMS, and the transcriptional pattern by DNA damage agent for RNRS mRNA. RNRS transcript is induced in response to a wide variety of agents that either damage D7A directly through chemical modification or induce stress by blocking DNA synthesis. As results, dpbll-1 cells are sensitive to DNA damage agents and the level of RNR3 mRNA is reduced approximately 40% than wild type cells. Moreover, we found the same results in dpb2-1 cells. Therefore, we propose that DPB2 and DPBll act as a sensor of replication that coordinates the transcriptional and cell cycle responses to replication blocks.

• Molecules and Cells
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• v.21 no.1
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• pp.63-75
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• 2006

The 5′-region of Potato virus X (PVX) RNA, which contains an AC-rich, single-stranded region and stem-loop structure 1 (SL1), affects RNA replication and assembly. Using Systemic Evolution of Ligands by EXponential enrichment (SELEX) and the electrophoretic mobility shift assay, we demonstrate that SL1 interacts specifically with tobacco protoplast protein extracts (S100). The 36 nucleotides that correspond to the top region of SL1, which comprises stem C, loop C, stem D, and the tetra loop (TL), were randomized and bound to the S100. Remarkably, the wild-type (wt) sequence was selected in the second round, and the number of wt sequences increased as selection proceeded. All of the selected clones from the fifth round contained the wt sequence. Secondary structure predictions (mFOLD) of the recovered sequences revealed relatively stable stem-loop structures that resembled SL1, although the nucleotide sequences therein were different. Moreover, many of the clones selected in the fourth round conserved the TL and C-C mismatch, which suggests the importance of these elements in host protein binding. The SELEX clone that closely resembled the wt SL1 structure with the TL and C-C mismatch was able to replicate and cause systemic symptoms in plants, while most of the other winners replicated poorly only on inoculated leaves. The RNA replication level on protoplasts was also similarly affected. Taken together, these results indicate that the SL1 of PVX interacts with host protein(s) that play important roles related to virus replication.

• Molecules and Cells
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• v.43 no.5
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• pp.469-478
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• 2020

Hepatitis C virus (HCV) propagation is highly dependent on cellular proteins. To identify the host factors involved in HCV propagation, we previously performed protein microarray assays and identified the LIM and SH3 domain protein 1 (LASP-1) as an HCV NS5A-interacting partner. LASP-1 plays an important role in the regulation of cell proliferation, migration, and protein-protein interactions. Alteration of LASP-1 expression has been implicated in hepatocellular carcinoma. However, the functional involvement of LASP-1 in HCV propagation and HCV-induced pathogenesis has not been elucidated. Here, we first verified the protein interaction of NS5A and LASP-1 by both in vitro pulldown and coimmunoprecipitation assays. We further showed that NS5A and LASP-1 were colocalized in the cytoplasm of HCV infected cells. NS5A interacted with LASP-1 through the proline motif in domain I of NS5A and the tryptophan residue in the SH3 domain of LASP-1. Knockdown of LASP1 increased HCV replication in both HCV-infected cells and HCV subgenomic replicon cells. LASP-1 negatively regulated viral propagation and thereby overexpression of LASP-1 decreased HCV replication. Moreover, HCV propagation was decreased by wild-type LASP-1 but not by an NS5A binding-defective mutant of LASP-1. We further demonstrated that LASP-1 was involved in the replication stage of the HCV life cycle. Importantly, LASP-1 expression levels were increased in persistently infected cells with HCV. These data suggest that HCV modulates LASP-1 via NS5A in order to regulate virion levels and maintain a persistent infection.

• Bulletin of the Korean Chemical Society
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• v.26 no.2
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• pp.285-291
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• 2005

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is the viral RNA-dependent RNA polymerase (RdRp), which is the essential catalytic enzyme for the viral replication and is an appealing target for the development of new therapeutic agents against HCV infection. A small amount of serum from a single patient with hepatitis C was used to get the genome of a Korean HCV isolate. Sequence analysis of NS5B 1701 nucleotides showed the genotype of a Korean isolate to be subtype 1b. The soluble recombinant HCV NS5B polymerase lacking the C-terminal 24 amino acids was expressed and purified to homogeneity. With the highly purified NS5B protein, we established in vitro systems for RdRp activity to identify potential polymerase inhibitors. The rhodanine family compounds were found to be potent and specific inhibitors of NS5B from high throughput screening (HTS) assay utilizing the scintillation proximity assay (SPA) system. The binding mode of an inhibitor was analyzed by measuring various kinetic parameters. Lineweaver-Burk plots of the inhibitor suggested it binds not to the active site of NS5B polymerase, but to an allosteric site of the enzyme. The activity of NS5B in in vitro polymerase reactions with homopolymeric RNA requires interaction with multiple substrates that include a template/primer and ribonucleotide triphosphate. Steady-state kinetic parameter, such as Km, was determined for the ribonucleotide triphosphate. One of compounds found interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitively with respect to UTP. Furthermore, we also investigated the ability of the compound to inhibit NS5B-directed viral RNA replication using the Huh7 cell-based HCV replicon system. The investigation is potentially very useful for the utility of such compounds as anti-hepatitic agents.