• Korean Journal of Veterinary Research
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• v.36 no.3
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• pp.627-633
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• 1996

To analyze the genomic diversity of transmissible gastroenteritis virus (TGEV), the N-terminal half of the spike (S) glycoprotein gene and nonstructural protein gene (open reading frames 3 and 3-1) were amplified by reverse transcriptase reaction and polymerase chain reaction (RT-PCR), and analyzed using restriction fragment length polymorphism (RFLP) patterns of the amplified DNA. In this study, TGEV Miller (M6) and Purdue (P115) strains were used as reference strains, and two vaccine strains (MSV and STC3) and four Korea isolates (P44, VRI-WP, VRI-41, and VRI-48) were analyzed. All TGEV strains were amplified with three TGEV primer pairs. Although there was some exception in RFLP analysis, this method differentiated TGEV strains into following groups : Miller group (M6 and MSV), Purdue group (PUS, STC3, P44, VRI-WP, VRI-41, and VRI-48). Using Sau3AI and SspI, VRI-48 was differentiated from the Miller and Purdue type viruses. The RT/PCR in conjuction with RFLP analysis was a rapid and valuable tool for differentiating several strains of TGEV. This study revealed the occurences of distinct difference in genome of TGEV strains.

• The Journal of Korean Society of Virology
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• v.29 no.4
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• pp.221-233
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• 1999

Prospect Hill Virus (PHV) is the well known serotype of hantavirus, a newly established genus in family Bunyaviridae. Extensive studies have upheld the original view of PHV genetics with three genes such as nucleocapsid (N) protein, envelope proteins (G1, G2) and RNA dependent RNA polymerase. In this study, we report the existence of additional gene that is encoded in an overlapping reading frame of the N protein gene within S genome segment of PHV. This gene is expected to encode a nonstructural small (NSs) protein and it seems to be only found in PHV infected cell. The presence and synthesis of NSs protein could be demonstrated in the cell infected with PHV using anti-peptide sera specific to the predicted amino acid sequence deduced from the second open reading frame. Ribosomal synthesis of this protein appears to occur at AUG codon at the 83rd base of S genome segment, downstream of N protein initiation codon. This protein is small in size (10.4 KDa) and highly basic in nature. The expression strategy of NSs protein appears that a signal mRNA is used to translate both N and NSs protein in PHV infected cell. 10 KDa protein in virus infected cell lysates can bind to mimic dsRNA. This fact strongly suggests that NSs protein may be involved in virus replication on late phase of viral life cycle.

• The Plant Pathology Journal
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• v.27 no.4
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• pp.310-314
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• 2011

Nonstructural protein 3 (NS3) encoded by RNA3 of Rice stripe virus (RSV), known to be a suppressor of gene silencing, was cloned and sequenced. The cloned NS3 gene is composed of 636 nucleotides encoding 211 deduced amino acids, and showed a high degree of similarity with the equivalent genes isolated from Korea, Japan and China. The NS3 gene promoted the enhancement of transient gene expression and suppressed transgene co-silencing. In the transient GFP expression via agroinfiltration, GFP expression was dramatically enhanced in terms of both protein yield and expression period in the presence of NS3. The highest accumulation of GFP protein reached to 6.8% of total soluble proteins, which corresponded to a two-fold increase compared to that obtained in the absence of NS3. In addition, NS3 significantly suppressed the initiation of GFP co-silencing induced by the additive GFP infiltration in GFP-transgenic Nicotiana benthamiana. The NS3 gene was also found to be a stronger suppressor than Cucumber mosaic virus 2b. These observations are believed to be derived from the strong suppressive effect of NS3 on gene silencing, and indicate that NS3 could be used as an effective enhancer for the rapid production of foreign proteins in plants.

• The Journal of Korean Society of Virology
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• v.30 no.1
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• pp.1-10
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• 2000

The nonstructural protein NSP4, encoded by gene 10 of rotavirus, has been shown to playa role in viral assembly and known to be an enterotoxin, causing diarrhea in mouse pups. NSP4 gene was cloned from CBNU-2 (virulent bovine rotavirus/diarrheic fecal sample) and CBNU-1 (cell-culture adapted bovine rotavirus/isolated from CBNU-2 and 75 times passaged on MA104 cells), respectively, by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequenced and compared. The sequence data indicated that the NSP4 genes of bovine rotavirus (BRV) were 751 bases in length and encoded one open reading frame of 175 amino acids beginning at base 42 and terminating at base 569. Differences in nucleotide sequence between CBNU-2 and CBNU-1 were observed at 6 positions (base 274, 296, 391, 394, 396 and 579). NSP4 gene of BRV exhibited a high degree of nucleotide (90% and 94%) and amino acid sequence (91% and 97%) homology with those of SA11 and UK but a low degree of nucleotide (77% and 79%) and amino acids sequence (81% and 85%) homology with those of Wa and OSU.

• Journal of Microbiology and Biotechnology
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• v.31 no.2
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• pp.226-232
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• 2021

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging phlebovirus of the Phenuiviridae family that has been circulating in the following Asian countries: Vietnam, Myanmar, Taiwan, China, Japan, and South Korea. Despite the increasing infection rates and relatively high mortality rate, there is limited information available regarding SFTSV pathogenesis. In addition, there are currently no vaccines or effective antiviral treatments available. Previous reports have shown that SFTSV suppresses the host immune response and its nonstructural proteins (NSs) function as an antagonist of type I interferon (IFN), whose induction is an essential part of the host defense system against viral infections. Given that SFTSV NSs suppress the innate immune response by inhibiting type I IFN, we investigated the mechanism utilized by SFTSV NSs to evade IFNmediated response. Our co-immunoprecipitation data suggest the interactions between NSs and retinoic acid inducible gene-I (RIG-I) or TANK binding kinase 1 (TBK1). Furthermore, confocal analysis indicates the ability of NSs to sequester RIG-I and related downstream molecules in the cytoplasmic structures called inclusion bodies (IBs). NSs are also capable of inhibiting TBK1-interferon regulatory factor 3 (IRF3) interaction, and therefore prevent the phosphorylation and nuclear translocation of IRF3 for the induction of type I IFN. The ability of SFTSV NSs to interact with and sequester TBK1 and IRF3 in IBs demonstrate an effective yet unique method utilized by SFTSV to evade and suppress host immunity.

• Korean Journal of Veterinary Research
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• v.45 no.2
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• pp.207-214
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• 2005

The nonstructural protein 4 (NSP4) of rotavirus encoded by gene 10, plays an important role in rotavirus pathogenicity. In this study, NSP4 gene of avian rotavirus (AvRV-1, AvRV-2) was analyzed and expressed using baculovirus expression system. The sequence data indicated that the NSP4 gene of AvRV-1 and AvRV-2 were 727 bases in length, encoded one open reading frame of 169 amino acids beginning at base 41 and terminating at base 550, and had two glycosylation sites. Nucleotide sequences of NSP4 gene of AvRV-1 and AvRV-2 exhibited a high degree of homology ($88.1{\pm}7.6%$) with avian rotaviruses, namely Ty1, Ty3 and PO-13. Phylogenetic analysis showed that AvRV-1 and AvRV-2 belonged to genotype NSP4[E], which is widely found in group A avian rotaviruses. The baculovirus-expressed NSP4 migrated at 20-28 kDa and reacted with NSP4-specific antiserum by FA and Western blot. Furthermore, it was found to be a glycoprotein by using tunicamycin, which is a specific inhibitor of N-linked glycosylation.

• Molecules and Cells
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• v.40 no.3
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• pp.202-210
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• 2017

The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus (HCV) RNA genome is a multifunctional phosphoprotein. To analyse the influence of NS5A on apoptosis, we established an Hep-NS5A cell line (HepG2 cells that stably express NS5A) and induced apoptosis using tumour necrosis factor $(TNF)-{\alpha}$. We utilised the MTT assay to detect cell viability, real-time quantitative polymerase chain reaction and Western blot to analyse gene and protein expression, and a luciferase reporter gene experiment to investigate the targeted regulatory relationship. Chromatin immunoprecipitation was used to identify the combination of $NF-{\kappa}B$ and miR-503. We found that overexpression of NS5A inhibited $TNF-{\alpha}$-induced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the $TNF-{\alpha}$ induced Hep-mock cells was significantly less than the viability of the $TNF-{\alpha}$ induced Hep-NS5A cells, which demonstrates that NS5A inhibited $TNF-{\alpha}$-induced HepG2 cell apoptosis. Under $TNF-{\alpha}$ treatment, miR-503 expression was decreased and cell viability and B-cell lymphoma 2 (bcl-2) expression were increased in the Hep-NS5A cells. Moreover, the luciferase reporter gene experiment verified that bcl-2 was a direct target of miR-503, NS5A inhibited $TNF{\alpha}$-induced $NF-{\kappa}B$ activation and $NF-{\kappa}B$ regulated miR-503 transcription by combining with the miR-503 promoter. After the Hep-NS5A cells were transfected with miR-503 mimics, the data indicated that the mimics could reverse $TNF-{\alpha}$-induced cell apoptosis and blc-2 expression. Collectively, our findings suggest a possible molecular mechanism that may contribute to HCV treatment in which NS5A inhibits $NF-{\kappa}B$ activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.77-77
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• 1995

Human immunodeficiency virus type 1 (HIV-1) contains several nonstructural genes which are required for the viral replication and disease pathogenesis. Among them, tat and nef genes encode an essential transactivator of HIV-1 LTR and a pluripotent protein which seems to be essential for the in vivo but not in vitro viral replication, respectively. We constructed two tat and n of defective HIV-1 and tested for their ability to replicate in several T cells. The defective viruses did not replicate in CD4$\^$+/ T cells, but rescued in the recombinant Jurkat-tat cell which also contains tat gene. The replication of tat and nef defective HIV-1 which expresses chloramphenicol acetyltransferase(CAT) gene was easily detected by a sensitive CAT assay. No revertant was identified during the passages of the mutant viruses for more than two months in Jurkat-tat cells. tat and n of defective HIV-1 could be used instead of wild type viruse for several purposes such as inhibitor screening and development of attenuated AIDS vaccine.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.213-218
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• 1999

Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plants. In addition, it has been suggested that, due to their solubility, they can play an important role in helping plants survive periods of osmotic stress. In order to study the effect of levan synthesis on plant growth, the coding region of the levansucrase gene, which was isolated from Zymomonas mobilis, was introduced into tobacco plants using Agrobacterium tumefaciens-mediated transformation. The presence of the levansucrase gene in transgenic plants was verified by genomic DNA gel blot analysis. RNA gel blot and immunoblot analyses showed an accumulation of the corresponding transcript and protein product of the bacterial levansucrase gene in transgenic plants. Furthermore, a thin layer chromatography analysis revealed that fructans were synthesized and deposited in transgenic tobacco plants. When $T_1$ seeds were germinated and grown under polyethylene glycol-mediated drought stress or cold stress, the transgenic seedlings displayed a substantially higher level of growth than that of untransformed plants. These results suggest that fructans may playa significant role in the tolerance of plants under osmotic stress.

• BMB Reports
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• v.38 no.2
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• pp.151-160
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• 2005

By a cDNA array representing 2308 signal transduction related genes, we studied the expression profiles of HeLa cells stably transfected by Hepatitis C virus nonstructural protein 4B (HCV-NS4B). The alterations of the expression of four genes were confirmed by real-time quantitative RT-PCR; and the aldo-keto reductase family 1, member C1 (AKR1C1) enzyme activity was detected in HCV-NS4B transiently transfected HeLa cells and Huh-7, a human hepatoma cell line. Of the 2,308 genes we examined, 34 were up-regulated and 56 were down-regulated. These 90 genes involved oncogenes, tumor suppressors, cell receptors, complements, adhesions, transcription and translation, cytoskeletion and cellular stress. The expression profiling suggested that multiple regulatory pathways were affected by HCV-NS4B directly or indirectly. And since these genes are related to carcinogenesis, host defense system and cell homeostatic mechanism, we can conclude that HCV-NS4B could play some important roles in the pathogenesis mechanism of HCV.