• Journal of Plant Biology
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• v.37 no.3
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• pp.349-355
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• 1994

Viral RNA was extracted from a purified orchid strain of tobacco mosaic virus (TMV-O) from Cymbidium "Grace Kelly". Polyadenylated viral RNAs were primed with Not I-oligo (dT) primer-adapter. First-strand cDNAs were reversely transcribed by Moloney murine leukaemia virus reverse transcriptase (RNAse H-), and then second-strand cDNAs were synthesized by RNase H and DNA polymerase I. The resulting double-stranded cDNAs were ligated into pSPORT1 vector and transformed into competent E. coli strain JM109 cells. The size of cDNAs within the recombinant plasmids was ranging from 0.9 to 3.9 kb. Among the selected clones, pTMO-0205 and -0210 covered the 3' half and the 5' half of the viral genomic RNA, respectively, which were covering more than 99% of the viral genemo size based on sequencing analysis. Two cDNA fragments which were 3.1 kb BamHI and NotI fragement released from pTMO-0.205 and 3.3 kb SalI and BamHI fragment released from pTMO-0210 were ligated with T4 DNA ligase. The clone was almost entire length, lacking only 31 nucleotides from the 5' terminus based on the sequencing result. This method was shown to be efficiently applicable to other plant viral gnomic RNA for the construction of cDNA.n of cDNA.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1744-1754
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• 2014

The hepatitis C virus (HCV) RNA genome is replicated by an RNA replicase complex (RC) consisting of cellular proteins and viral nonstructural (NS) proteins, including NS5B, an RNA-dependent RNA polymerase (RdRp) and key enzyme for viral RNA genome replication. The HCV RC is known to be associated with an intracellular membrane structure, but the cellular components of the RC and their roles in the formation of the HCV RC have not been well characterized. In this study, we took a proteomic approach to identify stomatin, a member of the integral proteins of lipid rafts, as a cellular protein interacting with HCV NS5B. Co-immunoprecipitation and co-localization studies confirmed the interaction between stomatin and NS5B. We demonstrated that the subcellular fraction containing viral NS proteins and stomatin displays RdRp activity. Membrane flotation assays with the HCV genome replication-competent subcellular fraction revealed that the HCV RdRp and stomatin are associated with the lipid raft-like domain of membranous structures. Stomatin silencing by RNA interference led to the release of NS5B from the detergent-resistant membrane, thereby inhibiting HCV replication in both HCV subgenomic replicon-harboring cells and HCV-infected cells. Our results identify stomatin as a cellular protein that plays a role in the formation of an enzymatically active HCV RC on a detergent-resistant membrane structure.

• Korean Journal of Veterinary Research
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• v.32 no.3
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• pp.389-398
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• 1992

Molecular cloning was carried out on the Danish strain of bovine viral diarrhea virus(BVDV) to construct strategy for the diagnostic tools and effective vaccine of BVD afterwards. A recombinant DNA clone(No. 29) was established successfully from cDNA for viral RNA tailed with adenine homopolymer at 3'-end. $^{32}P$-labeled DNA probes of 300~1,800bp fragments, originating from the clone 29, directed specific DNA-RNA hybridization results with BVDV RNA. Recombinant DNA of the clone 29 was about 5,200bp representing 41.6% of the full length of Danish strain's RNA, and restriction sites were recognized for EcoR I, Sst I, Hin d III and Pst I restriction enzymes in the DNA fragment.

• Journal of Pharmaceutical Investigation
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• v.40 no.spc
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• pp.119-129
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• 2010

The emergence of new biological drugs based on RNA interference (RNAi) technology has been one of the most attractive issues in the field of gene therapy for years. However, the use of siRNA therapeutics in clinical settings is still limited due to lack of appropriate delivery systems for the highly charged macromolecular drug. In this review, recent development of major non-viral siRNA delivery systems, including lipid, liposome, polymer, and peptide-based carriers, is to be summarized.

• BMB Reports
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• v.32 no.1
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• pp.82-85
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• 1999

The movement protein of cucumber mosaic virus (CMV) is required for cell-to-cell movement of viral RNA. The movement of viral RNA occurs through the plant intercellular connection, the plasmodesmata. The viral movement protein was known to be multi-functional. In this work, a series of deletion mutants of CMV movement protein gene were created to identify the functional domains. The mutated movement proteins were produced as inclusion body in E. coli, and purified and renatured. A polyclonal antibody was raised against the CMV-Kor strain (Korean isolate) movement protein expressed in E. coli. The ability of the truncated proteins to bind to ssRNA was assayed by UV cross-linking and gel retardation analyses. The results indicate that the domain between amino acids 118 and 160 of CMV movement protein is essential for ssRNA binding.

• Korean Journal of Microbiology
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• v.26 no.3
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• pp.155-161
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• 1988

Nucleotide sequence homology between bovine, simian, and porcine rotavirus was determined by the RNA:RNA hybridization technique. Single stranded RNA, prepared in vitro with EDTA activated endogeneous viral RNA polymerase, was hhbridized with tritium labeled bovine rotavirus genomic RNA. The heteroduplex RNA was treated with single stranded RNA specific ribonucleases and the RNase resistant hybrid RNA was precipitated, and collected by filtration on a filter paper. Seventy four percent RNA sequence homology between bovine and simian rotavirus and 8 percent RNA sequence homology between bovine and porcine rotavirus was confirmed by hybridization between tritium labeled single stranded RNA and viral genomic RNA.

• The Plant Pathology Journal
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• v.28 no.1
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• pp.40-48
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• 2012

$Potato$ $virus$ $X$ (PVX) replication is precisely regulated by regulatory viral sequences and by viral and/or host proteins. In a previous study, we identified a 54-kDa cellular tobacco protein that bound to a region within the first 46 nucleotides (nt) of the 5' non-translated region (NTR) of the viral genome. Optimal binding was dependent upon the presence of an ACCA sequence at nt 10-13. To identify host factors that bind to 5' NTR elements including AC-rich sequences as well as stemloop 1 (SL1), we used northwestern blotting and matrixassisted laser desorption/ionization time-of-flight mass spectrometry for peptide mass fingerprinting. We screened several host factors that might affect PVX replication and selected a candidate protein, $Nicotiana$ $tabacum$ WRKY transcription factor 1 (NtWRKY1). We used a $Tobacco$ $rattle$ $virus$ (TRV)-based virus-induced gene silencing (VIGS) system to investigate the role of NtWRKY1 in PVX replication. Silencing of $WRKY1$ in $Nicotiana$ $benthamiana$ caused lethal apical necrosis and allowed an increase in PVX RNA accumulation. This result could reflect the balancing of PVX accumulation in a systemic $N.$ $benthamiana$ host to maintain PVX survival and still produce a suitable appearance of mosaic and mottle symptoms. Our results suggest that PVX may recruit the WRKY transcription factor, which binds to the 5' NTR of viral genomic RNA and acts as a key regulator of viral infection.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.4
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• pp.489-498
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• 2018

Objective: Foot and mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are major diseases that interrupt porcine production. Because they are viral diseases, vaccinations are of only limited effectiveness in preventing outbreaks. To establish an alternative multi-resistant strategy against FMD virus (FMDV) and PRRS virus (PRRSV), the present study introduced two genetic modification techniques to porcine cells. Methods: First, cluster of differentiation 163 (CD163), the PRRSV viral receptor, was edited with the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 technique. The CD163 gene sequences of edited cells and control cells differed. Second, short hairpin RNA (shRNAs) were integrated into the cells. The shRNAs, targeting the 3D gene of FMDV and the open reading frame 7 (ORF7) gene of PRRSV, were transferred into fibroblasts. We also developed an in vitro shRNA verification method with a target gene expression vector. Results: shRNA activity was confirmed in vitro with vectors that expressed the 3D and ORF7 genes in the cells. Cells containing shRNAs showed lower transcript levels than cells with only the expression vectors. The shRNAs were integrated into CD163-edited cells to combine the two techniques, and the viral genes were suppressed in these cells. Conclusion: We established a multi-resistant strategy against viral diseases and an in vitro shRNA verification method.

• 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.

• Journal of Crop Science and Biotechnology
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• v.11 no.2
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• pp.127-134
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• 2008

Tobamoviruses are the major viral pathogens of tomato and bell pepper. The preliminary results showed that Acibenzolar-Smethyl(ASM; S-methylbenzo(1,2,3) thiadiazole-7-carbothiate) pre-treatment to tomato and tobacco plants reduces the concentration of Tomato mosaic tobamovirus(ToMV) and Tobacco mosaic tobamovirus(TMV) in tomato and bell pepper seedlings, respectively. Pre-treatment of the indicator plant(Nicotiana glutinosa) with the ASM followed by challenge inoculation with tobamoviruses produced a reduced number and size of local lesions(67 and 79% protection over control to TMV and ToMV inoculation, respectively). In order to understand the mechanism of resistance the gene expression profiles of antiviral genes was examined. RT-PCR products showed higher expression of two viral resistance genes viz., alternative oxidase(AOX) and RNA dependent RNA polymerase(RdRp) in the upper leaves of the ASM-treated tomato plants challenge inoculation with ToMV. Further, the viral concentration was also quantified in the upper leaves by reverse transcription PCR using specific primer for movement protein of ToMV, as well as ELISA by using antisera against tobamoviruses. The results provided additional evidence that ASM pre-treatment reduced the viral movement to upper leaves. The results suggest that expressions of viral resistance genes in the host are the key component in the resistance against ToMV in the inducer-treated tomato plants.