TY - JOUR
T1 - Interactions between p27 and p88 replicase proteins of Red clover necrotic mosaic virus play an essential role in viral RNA replication and suppression of RNA silencing via the 480-kDa viral replicase complex assembly
AU - Mine, Akira
AU - Hyodo, Kiwamu
AU - Takeda, Atsushi
AU - Kaido, Masanori
AU - Mise, Kazuyuki
AU - Okuno, Tetsuro
N1 - Funding Information:
The authors thank S. A. Lommel for the original RNA1 and RNA2 cDNA clones of RCNMV Australian strain, and D. C. Baulcombe for Nicotiana benthamiana line 16C. The authors are also grateful to H. Iwakawa for helpful discussion. This work was supported in part by a Grant-in-Aid for Scientific Research (A) ( 18208004 ) and by a Grant-in-Aid for Scientific Research (A) ( 22248002 ) from the Japan Society for the Promotion of Science .
PY - 2010/11/25
Y1 - 2010/11/25
N2 - Red clover necrotic mosaic virus (RCNMV), a positive-sense RNA virus with a bipartite genome, encodes p27 and p88 replicase proteins that are required for viral RNA replication and suppression of RNA silencing. In this study, we indentified domains in p27 and p88 responsible for their protein-protein interactions using in vitro pull-down assays with the purified recombinant proteins. Coimmunoprecipitation analysis in combination with blue-native polyacrylamide gel electrophoresis using mutated p27 proteins showed that both p27-p27 and p27-p88 interactions are essential for the formation of the 480-kDa complex, which has RCNMV-specific RNA-dependent RNA polymerase activity. Furthermore, we found a good correlation between the accumulated levels of the 480-kDa complex and replication levels and the suppression of RNA silencing activity. Our results indicate that interactions between RCNMV replicase proteins play an essential role in viral RNA replication and in suppressing RNA silencing via the 480-kDa replicase complex assembly.
AB - Red clover necrotic mosaic virus (RCNMV), a positive-sense RNA virus with a bipartite genome, encodes p27 and p88 replicase proteins that are required for viral RNA replication and suppression of RNA silencing. In this study, we indentified domains in p27 and p88 responsible for their protein-protein interactions using in vitro pull-down assays with the purified recombinant proteins. Coimmunoprecipitation analysis in combination with blue-native polyacrylamide gel electrophoresis using mutated p27 proteins showed that both p27-p27 and p27-p88 interactions are essential for the formation of the 480-kDa complex, which has RCNMV-specific RNA-dependent RNA polymerase activity. Furthermore, we found a good correlation between the accumulated levels of the 480-kDa complex and replication levels and the suppression of RNA silencing activity. Our results indicate that interactions between RCNMV replicase proteins play an essential role in viral RNA replication and in suppressing RNA silencing via the 480-kDa replicase complex assembly.
KW - Blue-native PAGE
KW - Coimmunoprecipitation analysis
KW - Dianthovirus
KW - In vitro translation and RNA synthesis
KW - Positive-sense RNA virus
KW - Protein-protein interaction
KW - RNA silencing suppression
KW - RNA-dependent RNA polymerase
KW - Tombusviridae
KW - Viral RNA replication
KW - Viral replicase complex
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U2 - 10.1016/j.virol.2010.07.038
DO - 10.1016/j.virol.2010.07.038
M3 - Article
C2 - 20828775
AN - SCOPUS:77957659703
SN - 0042-6822
VL - 407
SP - 213
EP - 224
JO - Virology
JF - Virology
IS - 2
ER -