• The Plant Pathology Journal
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• 제17권1호
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• pp.22-28
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• 2001

Barley yellow dwarf virus PAV (BYDV-PAV) has a 5.7-kb positive-sense single-stranded RNA genome that contains six open reading frames (ORFs). BYDV-PAV produces three subgenomic RNAs (sgRNAs). The largest of which encodes the coat, 17-kDa, and readthrough proteins from two initiation codons. To investigate the role of intergenic and 3'-proximal noncoding regions (NCRs) in coat protein (CP) expression and BYDV-PAV replication, a full-length infectious cDNA of the RNA genome of an Illinois isolate of BYDV-PAV was constructed downstream of the Cauliflower mosaic virus-35S promoter. Linear DNA molecules of these cDNAs were infectious, expressed the 22-kDa CP, and produced both genomic RNA sgRNAs in ratios similar to those observed in protoplasts inoculated with viral RNA. The portion of 5'NCR of sgRNA1 between ORFs 2 and 3 was not required for, but enhanced translation of CP from ORF3. Mutants containing deletions in the NCR downstream of ORF5 failed to replicate in oat protoplasts. These results indicate that an intact 3$^1$NCR is required for BYDV-PAV replication.

• Journal of Microbiology and Biotechnology
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• 제24권12호
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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.

• Animal cells and systems
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• 제1권2호
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• pp.363-370
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• 1997

RNase MRP is a ribonucleoprotein complex with a site-specific endonuclease activity. Its original substrate for cleavage is the small mitochondrial RNA near the mitochondrial DNA replication origin, thus it was proposed to generate the primer for mtDNA replication. Recently, it has been shown to have another substrate in the nucleus, such as pre-S.8S ribosomal RNA in nucleolus. The gene for the RNA component of RNase MRP (MRP RNA) was found to be encoded by the nucleus genome, suggesting an interesting intracellular trafficking of MRP RNA to both mitochondria and nucleolus after transcription in nucleus. In this study, genomic DNA encoding MRP RNA was microinjected into the nucleus of Xenopus oocytes, to analyze promoter regions involved in the transcription. It showed that the proximal sequence element and TATA box are important for basal level transcription; octamer motif and Sp1 binding sites are for elevated level transcription. Most of Xenopus MRP RNA was exported out to the cytoplasm following transcription in the nucleus. Utilizing various hybrid constructs, export of MRP RNA was found to be regulated by the promoter and the 5' half of the coding region of the gene. Interestingly, the transcription in nucleus seems to be coupled to the export of MRP RNA to cytoplasm. Intracellular transport of injected MRP RNA can be easily visualized by whole-mount in situ hybridization following microinjection; it also shows possible intra-nuclear sites for transcription and export of MRP RNA.

• The Plant Pathology Journal
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• 제28권1호
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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.

• BMB Reports
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• 제33권1호
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• pp.59-62
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• 2000

Hepatitis C virus (HCV) nonstructural 5B (NS5B) protein is an RNA-dependent RNA polymerase (RdRp). To determine whether it can contribute to viral replication by interaction with cellular proteins, the yeast two-hybrid screening system was employed to screen a human liver cDNA library. Using the HCV NS5B as a bait, we have isolated positive clones encoding a cellular protein. The NS5B interacting protein, 5BIP, is a novel cellular protein of 170 amino acids. Interaction of the HCV NS5B protein with 5BIP was confirmed by a protein-protein blotting assay. Recently, we have demonstrated that NS5B possesses an RdRp activity and thus it is possible that 5BIP, in association with NS5B, plays a role in HCV replication.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.178-178
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• 1994

Hepatitis B Virus (HBV) is a DNA virus with a 3.2kb partially double-stranded genome. The life cycle of the virus involves a reverse transcription of the greater than genome length 3.5kb mRNA. This pegenomic RNA contains all the genetic information encoded by the virus and functions as an intermediate in viral replication. Tumor suppressor p53 has previously been shown to interact with the X-gene product of the HBV, which led us to hypothesize that p53 may act as a negative regulator of HBV replication and the role of the X-gene product is to overcome the p53-mediated restriction. As a first step to prove the above hypothesis, we tested whether p53 represses the propagation of HBV in in vitro replication system. By transient cotransfection of the plasmid containing a complete copy of the HBV genome and/or the plasmid encoding p53, we found that the replication of HBV is specifically blocked by wild-type p53. The levels of HBV DNA, HBs Ag and HBc/e Ag secreted in cell culture media were dramatically reduced upon coexpresion of wild-type p53 but not by the coexpression of the mutants of p53 (G154V and R273L). Furthermore, levels of RNAs originated from HBV genome were repressed more than 10 fold by the cotransfection of the p53 encoding plasmid. These results clearly states that p53 is a nesative regulator of the HBV replication. Next, to addresss the mechanism by which p53 represses the HBV replication, we performed the transient transfection experiments employing the pregenomic/core promoter-CAT(Chloramphenicol Acetyl Transferase) construct as a reporter. Cotransfection of wild-type p53 but not the mutant p53 expression plasmids repressed the CAT activity more than 8 fold. Integrating the above results, we propose that p53 represses the replication of HBV specifically by the down-regulation of the pregenomic/core promoter, which results in the reduced DNA synthesis of HBV. Currently, the mechanism by which HBV overcomes the observed p53-mediated restriction of replication is tinder investigation.

• 미생물학회지
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• 제38권3호
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• pp.221-229
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• 2002

일본뇌염바이러스(Japanese encephalitis virus, JEV) Nakayama strain을 초기 multiplicity of infection 5.0으로 Vero세포에 감염하여 1년 이상 안정적으로 바이러스를 방출하는 지속감염(persistently-infected)세포주를 확립하였다. 지속감염 세포에서 지속적으로 방출되는 총 11 개의 Plaque 형태 변이바이러스(morphology mutants)클론을 확보하였다. 분리된 변이바이러스의 복제효율을 분석한 결과 생물학적 표현형과 복제효율은 유의하게 상관하였다. 변이바이러스 RNA 게놈 양 말단의 non-coding region 및 envelop 단백질의 ORF에서는 유의한 염기서열 변화가 관찰되지 않아 JEV 약독화에 새로운 인자가 추가로 관여할 가능성을 제시하였다. 변이바이러스에 감염된 신선한 Vero세포는 wild-type JEV의 일반적 감염성상과 다르게 대다수의 세포가 유의할 만한 세포병변현상을 나타내지 않았다. 감염된 Vero세포에서 wild-type JEV 및 large plaque을 형성하는 변이바이러스의 경우 mRNA와 함께 Bcl-2의 발현은 모두 유의하게 감소하였으며 p53은 뚜렷하게 증가하였다. 반면 small plaque을 형성하는 변이바이러스의 감염세포에서는 Bcl-2와 p53 모두 유의한 변화를 볼 수 없었다. 이상의 결과들과 함께, 감염된 Vero세포의 internucleosomal DNA fragmentation과DNA profile의 유형분석에 따르면 궁극적 인 세포병변효과의 변화는 변이바이러스의 복제효율과 더불어 p53에 비의존적인 apoptosis 수위의 전반적인 감소에 기인하는 것으로 판단된다.

• BMB Reports
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• 제37권3호
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• pp.383-385
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• 2004

RNA interference (RNAi) is a process of sequence-specific gene silencing, which is initiated by double-stranded RNA (dsRNA). RNAi may also serve as an antiviral system in vertebrates. This study describes the inhibition of herpesvirus-6B (HHV-6B) replication by short interference RNAs (siRNAs) that are targeted to the U38 sequence that encodes DNA polymerase. When virus-infected SupT1 cells were treated by siRNA, these cells blocked the cytopathic effect (CPE) and detected the HHV-6B antibody-negative in indirect immunofluorescence assays (IFA). Our result suggests that RNAi can efficiently block Herpesvirus-6B replication.

• 미생물학회지
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• 제34권4호
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• pp.248-253
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• 1998

Esherichia coli(E. coli) K12 균주를 숙주세포로 삼는 박테리오파아지인 N4는 single-stranded DNA에 결합하는 단백질인 N4SSB(bacteriophage N4-coded single-stranded DNA-binding protein) 단백질을 만든다. N4SSB 단백질은 N4 DNA replication 뿐만 아니라 late transcription과 N4 DNA recombination에도 필요한 여러 가지 기능을 가진 단백질이다. N4 late transcription은 숙주세포인 E. coli의 $E{\sigma}^{70}$ RNA polymerase에 의해서 수행이 되나 N4SSB 단백질을 반드시 필요로 하기 때문에 N4SSB 단백질이 생성될 때까지는 N4 late promoter로부터 RNA 합성이 일어나지 않는다. 본 연구에서는 N4SSB의 N4 DNA replication과 late transcription, 그리고 N4 DNA recombination에 필요한 영역(domain)을 알아내기 위해서 여러 가지 돌연변이형 N4SSB 단백질을 만들어 N4 DNA replication과 late transcription, 그리고 N4 DNA recombination의 3가지 작용에 대한 in vivo 활성을 조사 분석하였다. 그 결과 N4SSB 단백질의 C-말단기에 있는 7개의 아미노산이 N4SSB 단백질의 활성에 중요하다는 것을 알 수 있었다. 특히 C-말단기의 7개의 아미노산에는 세 개의 lysine이 포함되어 있는데 이 lysine이 N4SSB 단백질의 활성에 중요한 역할을 한다는 것이 제시되었다.

• Bulletin of the Korean Chemical Society
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• 제30권11호
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• pp.2626-2630
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• 2009

2'($\beta$)-Hydroxymethylated adenosine is a potent and selective inhibitor of hepatitis C virus (HCV) replication. It targets the RNA-dependent RNA polymerase of HCV, NS5B. Synthesis and antiviral evaluation of carbocyclic versions are described. The cyclopentene intermediate ($9\beta$) was successfully synthesized through sequential Johnson-Claisen orthoester rearrangement and ring-closing metathesis (RCM). Coupling of bases via a Pd(0) catalyst, selective dihydroxylation, and desilylation yielded the target nucleoside analogues. The compounds 17 and 18 were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line and showed moderate antiviral activity with toxicity up to 20.0 and 24.7 ${\mu}g/mL$, respectively.