• BMB Reports
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• v.28 no.1
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• pp.27-32
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• 1995

Polyclonal antibodies against human DNA-dependent RNA polymerase II (HPP II) were generated from chicken egg yolk after immunization with RNA polymerase II as an antigen. The antibodies from egg yolk (IgY) were purified and characterized. IgY showed a specificity against DNA-dependent RNA polymerase II, and was a polyclonal antibody against 12 subunits of polymerase II. An amount of 0.35 mg of IgY was obtained freman HPP II-Sepharose affinity column using 10 eggs from a chicken immunized against RNA polymerase II as an antigen. These antibodies can be used for isolating the genes for RNA polymerase II components, and for in vitro transcription assays using HP-RNA polymerase II.

• Journal of Plant Biology
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• v.38 no.2
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• pp.129-136
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• 1995

Complementary DNA of the genomic RNA of odontoglossum ringspot virus Cymbidium strain (ORSV-Cy) was synthesized from polyadenylated viral RNA and cloned. Selected clones containing the viral RNA-dependent RNA polymerase gene of the virus has been sequenced by automated sequencing system. The complete nucleotide sequence of an open reading frame is 1377 base pairs in length, and encodes a protein of 458 amino acids about 52, 334 D. The 52 kD protein of ORSV shares four sequence motifs characteristic of viral RNA-dependent RNA polymerase. Comparison of the ORSV 52 kD protein sequence with that of other five viruses in tobamovirus group showed 76.0 to 60.7% homologies at the amino acid level and the conservation of the four motifs betwen the viruses.

• BMB Reports
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• v.43 no.4
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• pp.284-290
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• 2010

Replication of positive-strand RNA virus is mediated by a virus-encoded RNA-dependent RNA polymerase (RdRp). To study the replication of Ectropis obliqua virus (EoV), a newly identified insect virus belonging to the family Iflaviradae, we expressed the RNA polymerase domain in Escherichia coli and purified it on a Ni-chelating HisTrap affinity column. It is demonstrated that EoV RdRp initiated RNA synthesis in a primer and poly (A)-dependent manner in vitro. Furthermore, the effect of primer concentration, temperature, metal ions ($Mg^{2+}$, $Mn^{2+}$, and $K^+$) on enzymatic activity were determined. Our study represented a first step towards understanding the mechanism of EoV replication.

• BMB Reports
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• v.39 no.5
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• pp.571-577
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• 2006

Dendrolimus punctatus tetravirus (DpTV) has been identified as a new member of the genus Omegatetravirus of the family Tetraviridae that may be related serologically to Nudaurelia capensis virus ($N{\omega}V$). To establish the function of DpTV RNA genome and to better understand the mechanism of viral replication, the putative RNA-dependent RNA polymerase (RdRp) domain has been cloned and expressed in Escherichia coli. The recombinant protein was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate viral RNA synthesis in a primer-independent manner but not by terminal nucleotidyle transferase activity in the presence of $Mg^{2+}$ and RNA template. Mutation of the GDD to GAA interferes with the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.

• Bulletin of the Korean Chemical Society
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• v.27 no.1
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• pp.59-64
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• 2006

Unlike other viral polymerases, HCV RNA-dependent RNA polymerase (RdRp) has not been successfully inhibited by nucleoside analogues presumably due to its strong substrate specificity for RNA. Thus, in order to understand the RNA-specificity of HCV RdRp, the structural characteristics of the active site was investigated. The hereto unknown 2-OH binding pocket at the active site of RdRp provides invaluable implication for the development of novel anti-HCV nucleoside analogues.

• Journal of Microbiology and Biotechnology
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• v.4 no.4
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• pp.264-269
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• 1994

To determine the nucleotide sequence of the ds RNA segment B containing the RNA dependent RNA polymerase (RdRp) gene of the DRT strain of infectious pancreatic necrosis virus (lPNV), the cDNA of the ds RNA segment B of the DRT strain of IPNV was synthesized using the reverse transcriptase (RT)-polymerase chain reaction (PCR) and its cDNA nucleotide sequence was determined. The DRT segment B was 2, 783 bp long and contained only a single long open reading frame (ORF) of 2, 535 bp in length. This ORF nucleotides encoded the VPl protein, the putative RdRp of IPNV. The VPl protein comsisted of 845 amino acids. The molecular weight of the RdRp, as deduced from the nucleotide sequence, is 94, 426. The nucleotide sequence of the ORF of the DRT showed 89.7% homology to the Jasper strain, but 80.8% to the Sp strain. The amino acid sequence of the ORF of the DRT sho.wed 97.6% homology to the Jasper strain, but 88.7% to the Sp strain. The conserved GTP-binding motif was detected in VPl protein.

• Journal of Microbiology
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• v.44 no.1
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• pp.11-22
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• 2006

Escherichia coli protein Rho is required for the factor-dependent transcription termination by an RNA polymerase and is essential for the viability of the cell. It is a homohexameric protein that recognizes and binds preferably to C-rich sites in the transcribed RNA. Once bound to RNA, it utilizes RNA-dependent ATPase activity and subsequently ATPase-dependent helicase activity to unwind RNA-DNA hybrids and release RNA from a transcribing elongation complex. Studies over the past few decades have highlighted Rho as a molecule and have revealed much of its mechanistic properties. The recently solved crystal structure could explain many of its physiological functions in terms of its structure. Despite all these efforts, many of the fundamental questions pertaining to Rho recognition sites, differential ATPase activity in response to different RNAs, translocation of Rho along the nascent transcript, interactions with elongation complex and finally unwinding and release of RNA remain obscure. In the present review we have attempted to summarize 'the knowns' and 'the unknowns' of the Rho protein revealed by the recent developments in this field. An attempt has also been made to understand the physiology of Rho in the light of its phylogeny.

• 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.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1634-1639
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• 2006

Hepatitis C virus (HCV)-encoded nonstructural protein 5B (NS5B) possesses RNA-dependent RNA polymerase activity, which is considered essential for viral proliferation. Thus, HCV NS5B is a good therapeutic target protein for the development of anti-HCV agents. In this study, we isolated two different kinds of nuclease-resistant RNA aptamers with 2'-fluoro pyrimidines against the HCV NS5B from a combinatorial RNA library with 40 nucleotide random sequences, using SELEX technology. The isolated RNA aptamers were observed to specifically and avidly bind the HCV NS5B with an apparent $K_d$ of 5 nM and 18 nM, respectively, in contrast with the original RNA library that hardly bound the target protein. Moreover, these aptamers could partially inhibit RNA synthesis of the HCV subgenomic replicon when transfected into Huh-7 hepatoma cell lines. These results suggest that the RNA aptamers selected in vitro could be useful not only as therapeutic agents of HCV infection but also as a powerful tool for the study of the HCV RNA-dependent RNA polymerase mechanism.

• The Journal of Korean Society of Virology
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• v.27 no.1
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• pp.1-8
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• 1997

Human caliciviruses (HuCVs) cause sporadic cases and outbreaks of acute gastroenteritis (AGE). Three major genogroups of HuCVs have been described including the Norwalk virus (NV)-, the Snow Mountain virus (SMA)-, and the Sapporo-genogroups. This study describes the detection and genetic variation of HuCVs from hospitalized infants with AGE in Korea by RT-PCR and sequencing. The cDNA fragments of 206 to 470bp corresponding to the region of 3 primer pairs (36/35, 35/51 or 3/51) in the polymerase region of NV were generated. Of 185 stools screened, 8% were positive by RT-PCR and their sequences showed that all strains contained the GLPSG and YGDD motifs which are conserved for HuCVs. Amino acid (aa) sequence analysis showed that these strains can be divided into 3 major genogroups. High conservation was observed in that one strain shares 100% of aa sequence with Southampton virus, another shares 99% with the Sapporo virus, and six strains share 90 to 95% with Snow Mountain virus. However, significant sequence variation was also found in other strains. This study indicates that all major genogroups of HuCVs are circulating in Korea.