徳島大学 教育・研究者情報データベース(EDB)

1. 徳島大学.疾患酵素学研究センター(2007年4月1日〜2016年3月31日/->組織[徳島大学.先端酵素学研究所.次世代酵素学研究領域])

1. (英) Mine Akira

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2. (英) Takeda Atsushi

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3. 谷口 貴子

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4. 谷口 寿章

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5. (英) Kaido Masanori

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6. (英) Mise Kazuyuki

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7. (英) Okuno Tetsuro

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(英) Identification and characterization of the 480-kilodalton template-specific RNA-dependent RNA polymerase complex of red clover necrotic mosaic virus.

(英) Replication of positive-strand RNA viruses occurs through the assembly of membrane-associated viral RNA replication complexes that include viral replicase proteins, viral RNA templates, and host proteins. Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA plant virus with a genome consisting of RNA1 and RNA2. The two proteins encoded by RNA1, a 27-kDa protein (p27) and an 88-kDa protein containing an RNA-dependent RNA polymerase (RdRP) motif (p88), are essential for RCNMV RNA replication. To analyze RCNMV RNA replication complexes, we used blue-native polyacrylamide gel electrophoresis (BN/PAGE), which enabled us to analyze detergent-solubilized large membrane protein complexes. p27 and p88 formed a complex of 480 kDa in RCNMV-infected plants. As a result of sucrose gradient sedimentation, the 480-kDa complex cofractionated with both endogenous template-bound and exogenous template-dependent RdRP activities. The amount of the 480-kDa complex corresponded to the activity of exogenous template-dependent RdRP, which produced RNA fragments by specifically recognizing the 3'-terminal core promoter sequences of RCNMV RNAs, but did not correspond to the activity of endogenous template-bound RdRP, which produced genome-sized RNAs without the addition of RNA templates. These results suggest that the 480-kDa complex contributes to template-dependent RdRP activities. We subjected those RdRP complexes to affinity purification and analyzed their components using two-dimensional BN/sodium dodecyl sulfate-PAGE (BN/SDS-PAGE) and mass spectrometry. The 480-kDa complex contained p27, p88, and possible host proteins, and the original affinity-purified RdRP preparation contained HSP70, HSP90, and several ribosomal proteins that were not detected in the 480-kDa complex. A model for the formation of RCNMV RNA replication complexes is proposed.

1. (英) Host-Parasite Interactions
2. (英) Macromolecular Substances
3. (英) Molecular Weight
4. (英) Plant Diseases
5. (英) RNA Replicase
6. (英) RNA, Viral
7. (英) Templates, Genetic
8. (英) Tombusviridae
9. (英) Viral Proteins
10. (英) Virus Replication

1. (英) Article.Affiliation: Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kitashirakawa, Kyoto 606-8502, Japan.

2. (英) Article.PublicationTypeList.PublicationType: Journal Article

3. (英) Article.PublicationTypeList.PublicationType: Research Support, Non-U.S. Gov't

4. (英) OtherID: PMC2876630