[Show abstract][Hide abstract] ABSTRACT: Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.
[Show abstract][Hide abstract] ABSTRACT: The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.
[Show abstract][Hide abstract] ABSTRACT: Many plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to a few viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes.
[Show abstract][Hide abstract] ABSTRACT: Although positive-strand RNA [(+)RNA] viruses have a limited coding capacity, they can replicate efficiently in host cells because of their ability to use host-derived proteins, membranes, lipids, and metabolites, and to rewire cellular trafficking pathways. Previously, we showed that a plant RNA virus, the Red clover necrotic mosaic virus (RCNMV), hijacked Arf1 and Sar1, which are small GTPases that regulate the biogenesis of COPI and COPII vesicles, respectively, for viral RNA replication. These small GTPases are relocated from appropriate subcellular compartments to the viral RNA replication sites by p27 replication protein, which raises the possibility that RCNMV interferes with the cellular secretory pathway. Here, we examined this possibility by using green fluorescent protein-fused rice SCAMP1 and Arabidopsis LRR84A as secretory pathway marker proteins and showed that p27 inhibited the trafficking of these proteins. RCNMV-mediated inhibition of the host secretion pathway and its possible impact on plant-virus interaction are discussed.
[Show abstract][Hide abstract] ABSTRACT: Replication of positive-strand RNA [(+)RNA] viruses proceeds through well-orchestrated actions of both viral and host factors. Remarkable features of eukaryotic (+)RNA virus replication include hijacking of host factors by viral components and remodeling of intracellular membranes to establish the viral replication factory, where viral RNA is synthesized. Here we review recent progress in our understanding of how (+)RNA plant viruses use host factors to create favorable environments for viral RNA replication.
Journal of General Plant Pathology 03/2014; 80(2). DOI:10.1007/s10327-014-0505-7 · 0.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic 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 the 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 COPI vesicles on Golgi membranes. Using in vitro pull-down 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.
Journal of Virology 10/2012; 87(1). DOI:10.1128/JVI.02383-12 · 4.44 Impact Factor
[Show abstract][Hide abstract] 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.
Journal of Virology 08/2012; 86(22):12091-104. DOI:10.1128/JVI.01659-12 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Positive-strand RNA viruses require host intracellular membranes for replicating their genomic RNAs. In this study, we determined the domains and critical amino acids in p27 of Red clover necrotic mosaic virus (RCNMV) required for its association with and targeting of ER membranes in Nicotiana benthamiana plants using a C-terminally GFP-fused and biologically functional p27. Confocal microscopy and membrane-flotation assays using an Agrobacterium-mediated expression system showed that a stretch of 20 amino acids in the N-terminal region of p27 is essential for the association of p27 with membranes. We identified the amino acids in this domain required for the association of p27 with membranes using alanine-scanning mutagenesis. We also found that this domain contains amino acids not critical for the membrane association but required for the formation of viral RNA replication complexes and negative-strand RNA synthesis. Our results extend our understanding of the multifunctional role of p27 in RCNMV replication.
[Show abstract][Hide abstract] ABSTRACT: The specific recognition of genomic RNAs by viral replicase proteins is a key regulatory step during the early replication process in positive-strand RNA viruses. In this study, we characterized the RNA-binding activity of the auxiliary replicase protein p27 of Red clover necrotic mosaic virus (RCNMV), which has a bipartite genome consisting of RNA1 and RNA2. Aptamer pull-down assays identified the amino acid residues of p27 involved in its specific interaction with RNA2. The RNA-binding activity of p27 correlated with its activity in recruiting RNA2 to membranes. We also identified the amino acids required for the formation of the 480-kDa replicase complex, a key player of RCNMV RNA replication. These amino acids are not involved in the functions of p27 that bind viral RNA or replicase proteins, suggesting an additional role for p27 in the assembly of the replicase complex. Our results demonstrate that p27 has multiple functions in RCNMV replication.
[Show abstract][Hide abstract] ABSTRACT: 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 identified 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.
[Show abstract][Hide abstract] ABSTRACT: Recognition of RNA templates by viral replicase proteins is one of the key steps in the replication process of all RNA viruses. However, the mechanisms underlying this phenomenon, including primary RNA elements that are recognized by the viral replicase proteins, are not well understood. Here, we used aptamer pulldown assays with membrane fractionation and protein-RNA coimmunoprecipitation in a cell-free viral translation/replication system to investigate how viral replicase proteins recognize the bipartite genomic RNAs of the Red clover necrotic mosaic virus (RCNMV). RCNMV replicase proteins bound specifically to a Y-shaped RNA element (YRE) located in the 3' untranslated region (UTR) of RNA2, which also interacted with the 480-kDa replicase complexes that contain viral and host proteins. The replicase-YRE interaction recruited RNA2 to the membrane fraction. Conversely, RNA1 fragments failed to interact with the replicase proteins supplied in trans. The results of protein-RNA coimmunoprecipitation assays suggest that RNA1 interacts with the replicase proteins coupled with their translation. Thus, the initial template recognition mechanisms employed by the replicase differ between RCNMV bipartite genomic RNAs and RNA elements are primary determinants of the differential replication mechanism.
Journal of Virology 10/2010; 85(1):497-509. DOI:10.1128/JVI.01754-10 · 4.44 Impact Factor