NDLI: Identification and Characterization of the 480-Kilodalton Template-Specific RNA-Dependent RNA Polymerase Complex of Red Clover Necrotic Mosaic Virus ▿

Content Provider	PubMed Central
Author	Mine, Akira Takeda, Atsushi Taniguchi, Takako Taniguchi, Hisaaki Kaido, Masanori Mise, Kazuyuki Okuno, Tetsuro
Copyright Year	2010
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.
Related Links	http://dx.doi.org/10.1128/jvi.00054-10
Ending Page	6081
Page Count	12
Starting Page	6070
File Format	PDF
ISSN	0022538X
e-ISSN	10985514
Journal	Journal of Virology
Issue Number	12
Volume Number	84
Language	English
Publisher	American Society for Microbiology (ASM)
Publisher Date	2010-06-15
Access Restriction	Open
Rights Holder	American Society for Microbiology (ASM)
Subject Keyword	Immunology Virology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Virology Immunology Microbiology Insect Science

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