title: Differential roles of Hsp70 and Hsp90 in the assembly of the replicase complex of a positive-strand RNA plant virus.
abstract: Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Mine
authors.forename: Kiwamu
authors.initials: K
authors.lastname: Hyodo
authors.forename: Yuri
authors.initials: Y
authors.lastname: Tajima
authors.forename: Kusumawaty
authors.initials: K
authors.lastname: Kusumanegara
authors.forename: Takako
authors.initials: T
authors.lastname: Taniguchi
authors.forename: Masanori
authors.initials: M
authors.lastname: Kaido
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Mise
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Taniguchi
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Okuno
mesh_terms: D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism; D020868:Gene Silencing; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D010944:Plants / Q000821:virology*; D011485:Protein Binding; D014176:Protein Biosynthesis; D012328:RNA Viruses / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics; D014026:Nicotiana / Q000821:virology; D019183:Tombusviridae / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase