title: Identification and characterization of the 480-kilodalton template-specific RNA-dependent RNA polymerase complex of red clover necrotic mosaic virus.
abstract: 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.
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kitashirakawa, Kyoto 606-8502, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Mine
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takeda
authors.forename: Takako
authors.initials: T
authors.lastname: Taniguchi
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Taniguchi
authors.forename: Masanori
authors.initials: M
authors.lastname: Kaido
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Mise
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Okuno
mesh_terms: D006790:Host-Parasite Interactions; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D010935:Plant Diseases / Q000821:virology; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D019183:Tombusviridae / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D046911:Macromolecular Substances; D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase