Japan bAssembly 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. Most plant and animal viruses are positive-strand RNA viruses, which have single-stranded messenger-sense genomic RNAs. These viruses often induce host membrane rearrangements to form organelle-like compartments in which viral genomic RNAs are replicated via negative-strand RNA intermediates by the viral replicase complexes (10). Viral replicase complexes comprise multiple proteins, including viral auxiliary proteins, viral RNAdependent RNA polymerase (RdRP), and host proteins (61). Viral replicase complexes have been studied extensively by characterizing their RdRP activities and the functions of the viral and host components of the complexes. These studies have provided important information about the mechanisms regulating genome replication (15, 19, 47, 89), viral pathogenicity (68, 69), and virushost interactions (24,25,32,33). However, an important question remains: how do multiple viral and host components assemble properly into the replicase complex?Molecular chaperones are essential for cell viability by ensuring folding of newly synthesized proteins, refolding of misfolded or aggregated proteins, protein complex assembly and disassembly, membrane translocation of organellar and secretory proteins, protein degradation, and activities of regulatory proteins in signal transduction pathways (12,18,51). In eukaryotic cells, the abundant and highly conserved molecular chaperones heat shock proteins Hsp70 and Hsp90 play central roles in the biological processes mentioned above, and the activities of Hsp70 and Hsp90 are modulated by a variety of cochaperones (37,80). Considering their pivotal roles i...

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOHderived ROS was demonstrated for the replication of another (+) RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.positive-strand RNA virus | viral RNA replication | reactive oxygen species | respiratory burst oxidase homolog | calcium-dependent protein kinase P lants have evolved complicated and sophisticated strategies to survive environmental changes. The rapid generation of reactive oxygen species (ROS) is one of the hallmarks of plant responses to various biotic and abiotic stresses (1). Plant NADPH oxidase, termed RBOHs (respiratory burst oxidase homologs), localize at the plasma membrane (PM) and intracellular compartments including the Golgi apparatus (2, 3) and play a key role in ROS production upon the perception of environmental stresses (4). In Nicotiana benthamiana, an RBOHB (NbRBOHB) plays important roles in ROS production triggered by microbe-associated molecular patterns (MAMPs), such as bacterial flagellin and fungal chitin, and facilitates plant immunity against biotrophic pathogens including an oomycete pathogen Phytophthola infestance and tobacco mosaic virus (5-8). RBOH activity is tightly and coordinately regulated posttranslationally [e.g., activation by Ca 2+ , phosphatidic acid (PA), G proteins, or protein kinases] (9-12). It has recently been shown that receptor-like cytoplasmic kinases and calciumdependent protein kinases (CDPKs) regulate RBOHs activity via direct phosphorylation or through modulating regulators of RBOHs during recognition of MAMPs via corresponding surface-loca...

bEukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery. E ukaryotic positive-strand RNA [(ϩ)RNA] viruses replicate their genomes using membrane-bound replicase complexes, which contain multiple viral and host components. A growing number of host proteins that affect viral RNA replication have been identified using genome-wide and proteomics analyses in several animal and plant viruses (1-13). These host proteins are involved in translation, template selection, and the assembly of the viral replication complex (VRC) on intracellular membranes, which serve as the site of viral RNA replication (14). However, the functions of host proteins remain largely unknown.The replication compartments of (ϩ)RNA viruses are derived from various cellular organelle membranes, such as the endoplasmic reticulum (ER), mitochondria, chloroplasts, peroxisomes, and the Golgi apparatus (15-17). The formation of viral replication compartments generally involves the emergence of spherules, vesicles, and multivesicular bodies associated with various organelles (15, 17). Although viral proteins play an essential role in the formation of replication compartments containing VRCs, host factors also regulate this process (14,15,18). Tomato bushy stunt virus (TBSV) coopts the proteins of the endosomal sorting compl...

Aphids (order Hemiptera) are important insect pests of crops and are also vectors of many plant viruses. However, little is known about aphid-infecting viruses, particularly their diversity and relationship to plant viruses. To investigate the aphid viromes, we performed deep sequencing analyses of the aphid transcriptomes from infested barley plants in a field in Japan. We discovered virus-like sequences related to nege/ kita-, flavi-, tombus-, phenui-, mononega-, narna-, chryso-, partiti-, and luteoviruses. Using RT-PCR and sequence analyses, we determined almost complete sequences of seven nege/kitavirus-like virus genomes; one of which was a variant of the Wuhan house centipede virus (WHCV-1). The other six seem to belong to four novel viruses distantly related to Wuhan insect virus 9 (WhIV-9) or Hubei nege-like virus 4 (HVLV-4). We designated the four viruses as barley aphid RNA virus 1 to 4 (BARV-1 to-4). Moreover, some nege/kitavirus-like sequences were found by searches on the transcriptome shotgun assembly (TSA) libraries of arthropods and plants. Phylogenetic analyses showed that BARV-1 forms a clade with WHCV-1 and HVLV-4, whereas BARV-2 to-4 clustered with WhIV-9 and an aphid virus, Aphis glycines virus 3. Both virus groups (tentatively designated as Centivirus and Aphiglyvirus, respectively), together with arthropod virus-like TSAs, fill the phylogenetic gaps between the negeviruses and kitaviruses lineages. We also characterized the flavi/jingmen-like and tombus-like virus sequences as well as other RNA viruses, including six putative novel viruses, designated as barley aphid RNA viruses 5 to 10. Interestingly, we also discovered that some aphid-associated viruses, including nege/kita-like viruses, were present in different aphid species, raising a speculation that these viruses might be distributed across different aphid species with plants being the reservoirs. This study provides novel information on the diversity and spread of nege/kitavirus-related viruses and other RNA viruses that are associated with aphids.

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