• Title, Summary, Keyword: RdRp

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Understanding the RNA-Specificity of HCV RdRp: Implications for Anti-HCV Drug Discovery

  • Kim, Jin-young;Chong, You-hoon
    • 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.

RNA silencing-mediated resistance is related to biotic / abiotic stresses and cellular RdRp expression in transgenic tobacco plants

  • Wu, Xiao-Liang;Hou, Wen-Cui;Wang, Mei-Mei;Zhu, Xiao-Ping;Li, Fang;Zhang, Jie-Dao;Li, Xin-Zheng;Guo, Xing-Qi
    • BMB Reports
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    • v.41 no.5
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    • pp.376-381
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    • 2008
  • The discovery of RNA silencing inhibition by virus encoded suppressors or low temperature leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRp) has been previously characterized to be essential for transgene-mediated RNA silencing. Here we showed that low temperature led to the inhibition of RNA silencing, the loss of viral resistance and the reduced expression of host RdRp homolog (NtRdRP1) in transgenic T4 progeny with untranslatable potato virus Y coat protein (PVY-CP) gene. Moreover, RNA silencing and the associated resistance were differently inhibited by potato virus X (PVX) and tobacco mosaic virus (TMV) infections. The increased expression of NtRdRP1 in both PVX and TMV infected plants indicated its general role in response to viral pathogens. Collectively, we propose that biotic and abiotic stress factors affect RNA silencing-mediated resistance in transgenic tobacco plants and that their effects target different steps of RNA silencing.

The First Identified Citrus tristeza virus Isolate of Turkey Contains a Mixture of Mild and Severe Strains

  • Cevik, Bayram;Yardimci, Nejla;Korkmaz, Sava
    • The Plant Pathology Journal
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    • v.29 no.1
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    • pp.31-41
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    • 2013
  • The presence of Citrus tristeza virus (CTV) has previously been reported in citrus growing regions of Turkey. All serologically and biologically characterized isolates including I$\breve{g}$d${\i}$r, which was the first identified CTV isolates from Turkey, were considered mild isolates. In this study, molecular characteristics of the I d r isolate were determined by different methods. Analysis of the I$\breve{g}$d${\i}$r isolate by western blot and BD-RT-PCR assays showed the presence of MCA13 epitope, predominantly found in severe isolates, in the I$\breve{g}$d${\i}$r isolate revealing that it contains a severe component. For further characterization, the coat protein (CP) and the RNA-depen-dent RNA polymerase (RdRp) genes representing the 3' and 5' half of CTV genome, respectively, were amplified from dsRNA by RT-PCR. Both genes were cloned separately and two clones for each gene were sequenced. Comparisons of nucleotide and deduced amino acid sequences showed that while two CP gene sequences were identical, two RdRp clones showed only 90% and 91% sequence identity in their nucleotide and amino acid sequences, respectively, suggesting a mixed infection with different strains. Phylogenetic analyses of the CP and RdRp genes of I$\breve{g}$d${\i}$r isolate with previously characterized CTV isolates from different citrus growing regions showed that the CP gene was clustered with NZRB-TH30, a resistance breaking isolate from New Zealand, clearly showing the presence of severe component. Furthermore, two different clones of the RdRp gene were clustered separately with different CTV isolates with a diverse biological activity. While the RdRp-1 was clustered with T30 and T385, two well-characterized mild isolates from Florida and Spain, respectively, the RdRp-2 was most closely related to NZRB-G90 and NZRB-TH30, two well-characterized resistance breaking and stem pitting (SP) isolates from New Zealand confirming the mixed infection. These results clearly demonstrated that the I$\breve{g}$d${\i}$r isolate, which was previously described as biologically a mild isolate, actually contains a mixture of mild and severe strains.

Nucleotide Sequence Analysis of the RNA-dependent RNA Polymerase Gene of Infectious Pancreatic Necrosis Virus DRT Strain

  • Lee, Hyung-Hoan;Chung, Hye-Kyung;Lee, Seong-Hun
    • 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.

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Double-stranded RNA virus in Korean Isolate IH-2 of Trichomonas vaginalis

  • Kim, Jong-Wook;Chung, Pyung-Rim;Hwang, Myung-Ki;Choi, Eun-Young
    • The Korean Journal of Parasitology
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    • v.45 no.2
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    • pp.87-94
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    • 2007
  • In this study, we describe Korean isolates of Trichomonas vaginalis infected with double-stranded (ds) RNA virus (TVV). One T. vaginalis isolate infected with TVV IH-2 evidenced weak pathogenicity in the mouse assay coupled with the persistent presence of a dsRNA, thereby indicating a hypovirulence effect of dsRNA in T. vaginalis. Cloning and sequence analysis results revealed that the genomic dsRNA of TVV IH-2 was 4,647 bp in length and evidenced a sequence identity of 80% with the previously-described TVV 1-1 and 1-5, but only a 42% identity with TVV 2-1 and 3 isolates. It harbored 2 overlapping open reading frames of the putative capsid protein and dsRNA-dependent RNA polymerase (RdRp). As previously observed in the TVV isolates 1-1 and 1-5, a conserved ribosomal slip-page heptamer (CCUUUUU) and its surrounding sequence context within the consensus 14-nt overlap implied the gene expression of a capsid protein-RdRp fusion protein, occurring as the result of a potential ribosomal frameshift event. The phylogenetic analysis of RdRp showed that the Korean TVV If-2 isolate formed a compact group with TVV 1-1 and 1-5 isolates, which was divergent from TVV 2-1, 3 and other viral isolates classified as members of the Giardiavirus genus.

Electrophysiological and Histologic Evaluation of the Time Course of Retinal Degeneration in the rd10 Mouse Model of Retinitis Pigmentosa

  • Jae, Seol A;Ahn, Kun No;Kim, Ji Young;Seo, Je Hoon;Kim, Hyong Kyu;Goo, Yong Sook
    • The Korean Journal of Physiology and Pharmacology
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    • v.17 no.3
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    • pp.229-235
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    • 2013
  • Among several animal models of retinitis pigmentosa (RP), the more recently developed rd10 mouse with later onset and slower rate of retinal degeneration than rd1 mouse is a more suitable model for testing therapeutic modalities. We therefore investigated the time course of retinal degeneration in rd10 mice before adopting this model in our interventional studies. Electroretinogram (ERG) recordings were carried out in postnatal weeks (PW) 3~5 rd10 (n=23) and wild-type (wt) mice (n=26). We compared the amplitude and implicit time of the b-wave of ERG records from wt and rd10 mice. Our results showed that b-wave amplitudes in rd10 mice were significantly lower and the implicit time of b-waves in rd10 mice were also significantly slower than that in wt mice ($20{\sim}160{\mu}V$ vs. $350{\sim}480{\mu}V$; 55~75 ms vs. 100~150 ms: p<0.001) through PW3 to PW5. The most drastic changes in ERG amplitudes and latencies were observed during PW3 to PW4. In multichannel recording of rd10 retina in PW2 to PW4.5, we found no significant difference in mean spike frequency, but the frequency of power spectral peak of local field potential at PW3 and PW3.5 is significantly different among other age groups (p<0.05). Histologic examination of rd10 retinae showed significant decrease in thickness of the outer nuclear layer at PW3. TUNEL positive cells were most frequently observed at PW3. From these data, we confirm that in the rd10 mouse, the most precipitous retinal degeneration occurs between PW3~PW4 and that photoreceptor degeneration is complete by PW5.

Identification of a Cellular Protein Interacting with RNA Polymerase of Hepatitis C Virus

  • Park, Kyu-Jin;Choi, Soo-Ho;Koh, Moon-Soo;Kim, Sung-Wan;Hwang, Soon-Bong
    • BMB Reports
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    • v.33 no.1
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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.

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Genome Sequence of Spinach Cryptic Virus 1, a New Member of the Genus Alphapartitivirus (Family Partitiviridae), Identified in Spinach

  • Park, Dongbin;Hahn, Yoonsoo
    • Journal of Microbiology and Biotechnology
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    • v.27 no.4
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    • pp.834-837
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    • 2017
  • A distinct double-stranded RNA (dsRNA) cryptic virus, named spinach cryptic virus 1 (SpCV1), was identified from spinach transcriptome datasets. The SpCV1 genome has two dsRNA genome segments. The larger dsRNA1 has an open reading frame for a conserved RNA-dependent RNA polymerase (RdRp). The smaller dsRNA2 encodes a putative coat protein (CP). The sequence identity of SpCV1 RdRp and CP to the closest cryptic virus is 81% and 60%, respectively. Phylogenetic analysis indicates that SpCV1 is a novel member of the genus Alphapartitivirus (family Partitiviridae).

Expression and characterization of RNA-dependent RNA polymerase of Ectropis obliqua virus

  • Lin, Meijuan;Ye, Shan;Xiong, Yi;Cai, Dawei;Zhang, Jiamin;Hu, Yuanyang
    • 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.

Molecular Characterization of a Novel Putative Partitivirus Infecting Cytospora sacchari, a Plant Pathogenic Fungus

  • Peyambari, Mahtab;Habibi, Mina Koohi;Fotouhifar, Khalil-Berdi;Dizadji, Akbar;Roossinck, Marilyn J.
    • The Plant Pathology Journal
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    • v.30 no.2
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    • pp.151-158
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    • 2014
  • Three double-stranded RNAs (dsRNAs), approximately 1.85, 1.65 and 1.27 kb in size, were detected in an isolate of Cytospora sacchari from Iran. Partial nucleotide sequence revealed a 1,284 bp segment containing one ORF that potentially encodes a 405 aa protein. This protein contains conserved motifs related to RNA dependent RNA polymerases (RdRp) that showed similarity to RdRps of partitiviruses. The results indicate that these dsRNAs represent a novel Partitivirus that we tentatively designate Cytospora sacchari partitivirus (CsPV). Treatment of the fungal strain by cyclohexamide and also hyphal tip culture had no effect on removing the putative virus. Phylogenetic analysis of putative RdRp of CsPV and other partitiviruses places CsPV as a member of the genus Partitivirus in the family Partitiviridae, and clustering with Aspergillus ochraceous virus 1.