Posttranscriptional gene silencing is mediated by RNA‐induced silencing complexes (RISCs) that contain AGO proteins and single‐stranded small RNAs. The assembly of plant AGO1‐containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90‐dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex‐bound AGO1. Moreover, CYP40 that was synthesized by in‐vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin‐binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90–AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants.

The cum1 and cum2 mutations of Arabidopsis thaliana inhibit cucumber mosaic virus (CMV) multiplication. In cum1 and cum2 protoplasts, CMV RNA and the coat protein accumulated to wild-type levels, but the accumulation of the 3a protein of CMV, which is necessary for cell-to-cell movement of the virus, was strongly reduced compared with that in wild-type protoplasts. In cum2 protoplasts, the accumulation of turnip crinkle virus (TCV)-related RNA and proteins was also reduced. Positional cloning demonstrated that CUM1 and CUM2 encode eukaryotic translation initiation factors 4E and 4G, respectively. Unlike most cellular mRNA, the CMV RNA lacks a poly(A) tail, whereas the TCV RNA lacks both a 5′-terminal cap and a poly(A) tail. In vivo translation analyses, using chimeric luciferase mRNA carrying the terminal structures and untranslated sequences of the CMV or TCV RNA, demonstrated that these viral untranslated sequences contain elements that regulate the expression of encoded proteins positively or negatively. The cum1 and cum2 mutations had different effects on the action of these elements, suggesting that the cum1 and cum2 mutations cause inefficient production of CMV 3a protein and that the cum2 mutation affects the production of TCV-encoded proteins.

The tomato Tm-1 gene confers resistance to tomato mosaic virus (ToMV). Here, we report that the extracts of Tm-1 tomato cells (GCR237) have properties that inhibit the in vitro RNA replication of WT ToMV more strongly than that of the Tm-1 -resistance-breaking mutant of ToMV, LT1. We purified this inhibitory activity and identified a polypeptide of ≈80 kDa (p80 ^GCR237 ) using LC–tandem MS. The amino acid sequence of p80 ^GCR237 had no similarity to any characterized proteins. The p80 ^GCR237 gene cosegregated with Tm-1 ; transgenic expression of p80 ^GCR237 conferred resistance to ToMV within tomato plants; and the knockdown of p80 ^GCR237 sensitized Tm-1 tomato plants to ToMV, indicating that Tm-1 encodes p80 ^GCR237 itself. We further show that in vitro -synthesized Tm-1 (p80 ^GCR237 ) protein binds to the replication proteins of WT ToMV and inhibits their function at a step before, but not after, the viral replication complex is formed on the membrane surfaces. Such binding was not observed for the replication proteins of LT1. These results suggest that Tm-1 (p80 ^GCR237 ) inhibits the replication of WT ToMV RNA through binding to the replication proteins.

Tobacco mosaic virus and other tobamoviruses have served as models for studying the mechanisms of viral RNA replication. In tobamoviruses, genomic RNA replication occurs via several steps: (a) synthesis of viral replication proteins by translation of the genomic RNA; (b) translation-coupled binding of the replication proteins to a 5′-terminal region of the genomic RNA; (c) recruitment of the genomic RNA by replication proteins onto membranes and formation of a complex with host proteins TOM1 and ARL8; (d) synthesis of complementary (negative-strand) RNA in the complex; and (e) synthesis of progeny genomic RNA. This article reviews current knowledge on tobamovirus RNA replication, particularly regarding how the genomic RNA is specifically selected as a replication template and how the replication proteins are activated. We also focus on the roles of the replication proteins in evading or suppressing host defense systems.

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.

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. Many important pathogens of plants, animals, and humans are positive-strand RNA viruses. They replicate via complementary RNA in replication complexes formed on host intracellular membranes. In the replication process, not only viral replication proteins but also host factors play important roles. Although many host factors whose knockdown affects the multiplication of positive-strand RNA viruses have been identified, the function of each host factor in virus multiplication is only poorly understood in most instances. In this paper, we show that a host small GTP-binding protein ARL8 is required for the multiplication of Tomato mosaic virus (ToMV), and that it forms a complex with ToMV replication proteins and another essential host factor TOM1 that is a seven-pass transmembrane protein. We further demonstrate that the replication proteins acquire the ability to synthesize negative-strand ToMV RNA and RNA 5′ cap only in the presence of both TOM1 and ARL8. The replication proteins of ToMV are multifunctional proteins that participate in RNA replication on membranes and RNA silencing suppression in the cytosol. Our results suggest that ToMV replication proteins are programmed to express their replication-related activities only on membranes through interactions with these host membrane proteins.