RRC ID 12785
Author Nishikiori M, Mori M, Dohi K, Okamura H, Katoh E, Naito S, Meshi T, Ishikawa M.
Title A host small GTP-binding protein ARL8 plays crucial roles in tobamovirus RNA replication.
Journal PLoS Pathog.
Abstract Tomato mosaic virus (ToMV), like other eukaryotic positive-strand RNA viruses, replicates its genomic RNA in replication complexes formed on intracellular membranes. Previous studies showed that a host seven-pass transmembrane protein TOM1 is necessary for efficient ToMV multiplication. Here, we show that a small GTP-binding protein ARL8, along with TOM1, is co-purified with a FLAG epitope-tagged ToMV 180K replication protein from solubilized membranes of ToMV-infected tobacco (Nicotiana tabacum) cells. When solubilized membranes of ToMV-infected tobacco cells that expressed FLAG-tagged ARL8 were subjected to immunopurification with anti-FLAG antibody, ToMV 130K and 180K replication proteins and TOM1 were co-purified and the purified fraction showed RNA-dependent RNA polymerase activity that transcribed ToMV RNA. From uninfected cells, TOM1 co-purified with FLAG-tagged ARL8 less efficiently, suggesting that a complex containing ToMV replication proteins, TOM1, and ARL8 are formed on membranes in infected cells. In Arabidopsis thaliana, ARL8 consists of four family members. Simultaneous mutations in two specific ARL8 genes completely inhibited tobamovirus multiplication. In an in vitro ToMV RNA translation-replication system, the lack of either TOM1 or ARL8 proteins inhibited the production of replicative-form RNA, indicating that TOM1 and ARL8 are required for efficient negative-strand RNA synthesis. When ToMV 130K protein was co-expressed with TOM1 and ARL8 in yeast, RNA 5'-capping activity was detected in the membrane fraction. This activity was undetectable or very weak when the 130K protein was expressed alone or with either TOM1 or ARL8. Taken together, these results suggest that TOM1 and ARL8 are components of ToMV RNA replication complexes and play crucial roles in a process toward activation of the replication proteins' RNA synthesizing and capping functions.
Volume 7(12)
Pages e1002409
Published 2011-12
DOI 10.1371/journal.ppat.1002409
PII PPATHOGENS-D-11-01529
PMID 22174675
PMC PMC3234234
MeSH Cell Line Chromatography, Liquid GTP-Binding Proteins / metabolism* Host-Parasite Interactions / genetics* Plant Proteins / genetics Plant Proteins / metabolism* Plants, Genetically Modified RNA, Viral / genetics Reverse Transcriptase Polymerase Chain Reaction Tandem Mass Spectrometry Tobacco / virology* Tobacco Mosaic Virus / metabolism* Viral Proteins / genetics Viral Proteins / metabolism* Virus Replication / genetics
IF 6.158
Times Cited 23
WOS Category PARASITOLOGY MICROBIOLOGY VIROLOGY
Resource
Arabidopsis / Cultured plant cells, genes pst12596