Host specific preference for low temperature in the multiplication of a tombusvirus, gentian virus A.

Gentian virus A (GeVA), a novel tombusvirus isolated from Japanese gentian, has shown only a limited ability to infect Japanese gentians under experimental conditions. In this study, temperature was found to affect the efficient multiplication of GeVA in Japanese gentians. GeVA efficiently multiplied in inoculated leaves of gentians at 18 °C but not at 23 °C. This low-temperature (18 °C)-preferred GeVA multiplication was specifically observed in Japanese gentians and Arabidopsis thaliana but not in other experimental plants, including Nicotiana benthamiana. In A. thaliana, visible defense responses, including pathogenesis-related protein 1 expression, were not detected at 23 °C. Furthermore, several A. thaliana mutants, including those defective in RNA silencing, with altered plant immunities did not allow GeVA to multiply to detectable levels at 23 °C. Taken together, these data suggest that unique interaction between GeVA and gentians/A. thaliana, which is independent of RNA silencing, may underlie the low-temperature-preferred multiplication of GeVA.

5'-Terminal stem-loop of carnation ringspot virus RNA1 is required for the efficient amplification of viral RNAs.

Carnation ringspot virus (CRSV) is the prototype virus of the genus Dianthovirus. Full-length cDNAs of CRSV strainsPV-0097 and PV-21 were constructed and the infectivity of in vitro transcripts was analyzed. Infectivity of PV-0097 and PV-21 to several plants was markedly higher than that of 1.30, a previously reported infectious CRSV clone. Overall RNA sequences of these viruses were similar, but PV-0097 and PV-21 contained additional nucleotides at the 5' end of RNA1. Stem-loop structures were predicted in the 5'-terminal region of PV-0097 and PV-21 RNA1 but not in 1.30 RNA1. Mutant CRSV 1.30 RNA1 that contains the terminal 4 nucleotides of PV-0097, predicted to fold a 5'-terminal stem-loop structure, recovered higher level accumulation of viral RNAs in the inoculated protoplasts and leaves of Nicotiana benthamiana. These results suggest that the 5'-terminal stem-loop structure of CRSV RNA1 plays an important role in efficient amplification of the virus.

Inappropriate Expression of an NLP Effector in Colletotrichum orbiculare Impairs Infection on Cucurbitaceae Cultivars via Plant Recognition of the C-Terminal Region.

The hemibiotrophic pathogen Colletotrichum orbiculare preferentially expresses a necrosis and ethylene-inducing peptide 1 (Nep1)-like protein named NLP1 during the switch to necrotrophy. Here, we report that the constitutive expression of NLP1 in C. orbiculare blocks pathogen infection in multiple Cucurbitaceae cultivars via their enhanced defense responses. NLP1 has a cytotoxic activity that induces cell death in Nicotiana benthamiana. However, C. orbiculare transgenic lines constitutively expressing a mutant NLP1 lacking the cytotoxic activity still failed to infect cucumber, indicating no clear relationship between cytotoxic activity and the NLP1-dependent enhanced defense. NLP1 also possesses the microbe-associated molecular pattern (MAMP) sequence called nlp24, recognized by Arabidopsis thaliana at its central region, similar to NLPs of other pathogens. Surprisingly, inappropriate expression of a mutant NLP1 lacking the MAMP signature is also effective for blocking pathogen infection, uncoupling the infection block from the corresponding MAMP. Notably, the deletion analyses of NLP1 suggested that the C-terminal region of NLP1 is critical to enhance defense in cucumber. The expression of mCherry fused with the C-terminal 32 amino acids of NLP1 was enough to trigger the defense of cucurbits, revealing that the C-terminal region of the NLP1 protein is recognized by cucurbits and, then, terminates C. orbiculare infection.

Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus.

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 PLDß. 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.

GAPDH--a recruits a plant virus movement protein to cortical virus replication complexes to facilitate viral cell-to-cell movement.

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.

ADP ribosylation factor 1 plays an essential role in the replication of a plant RNA virus.

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 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.

Differential roles of Hsp70 and Hsp90 in the assembly of the replicase complex of a positive-strand RNA plant virus.

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.

Identification of domains in p27 auxiliary replicase protein essential for its association with the endoplasmic reticulum membranes in Red clover necrotic mosaic virus.

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.

Viral cell-to-cell movement requires formation of cortical punctate structures containing Red clover necrotic mosaic virus movement protein.

Movement protein (MP) of Red clover necrotic mosaic virus (RCNMV) forms punctate structures on the cortical endoplasmic reticulum (ER) of Nicotiana benthamiana cells, which are associated with viral RNA1 replication (Kaido et al., Virology 395, 232-242. 2009). We investigated the significance of ER-targeting by MP during virus movement from cell to cell, by analyzing the function of a series of MPs with varying length deletions at their C-terminus, either fused or not fused with green fluorescent protein (GFP). The C-terminal 70 amino acids were crucial to ER-localization of MP-GFP and cell-to-cell movement of the recombinant virus encoding it. However, C-terminal deletion did not affect MP functions, such as increasing the size exclusion limit of plasmodesmata, single-stranded RNA binding in vitro, and MP interacting in vivo. We discuss the possible role of this MP region in virus movement from cell to cell.

Template recognition mechanisms by replicase proteins differ between bipartite positive-strand genomic RNAs of a plant virus.

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.

Interactions between p27 and p88 replicase proteins of Red clover necrotic mosaic virus play an essential role in viral RNA replication and suppression of RNA silencing via the 480-kDa viral replicase complex assembly.

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.

Endoplasmic reticulum targeting of the Red clover necrotic mosaic virus movement protein is associated with the replication of viral RNA1 but not that of RNA2.

Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA virus with a bipartite genome. The movement protein (MP) encoded by RNA2 is essential for viral movement. To obtain further insights into the viral movement mechanism, subcellular localizations of RCNMV MP fused with green fluorescent protein (MP:GFP) were examined in Nicotiana benthamiana epidermal cells and protoplasts. The MP:GFP expressed from the recombinant virus first appeared in the cell wall and subsequently was observed on the cortical endoplasmic reticulum (ER) as punctate spots. In contrast, the MP:GFP expressed transiently in the absence of other viral components was localized exclusively in the cell wall. Transient expression of the MP:GFP with a variety of RCNMV components revealed that the ER localization of the MP:GFP was associated with RNA1 replication, or its negative-strand RNA synthesis, but not those of RNA2 or replicase proteins per se. A model of RCNMV cell-to-cell movement is discussed.

Melandrium yellow fleck bromovirus infects Arabidopsis thaliana and has genomic RNA sequence characteristics that are unique among bromoviruses.

Melandrium yellow fleck bromovirus (MYFV) systemically infected Arabidopsis thaliana, although the susceptibility of several A. thaliana accessions to MYFV differed from their susceptibility to the other two bromoviruses infecting A. thaliana. We constructed full-length cDNA clones of MYFV genomic RNAs 1, 2, and 3 and determined their complete nucleotide sequences. Similar to Broad bean mottle bromovirus, (1) the 5'-terminal nucleotide of the MYFV genomic RNAs was adenine, and (2) the "D-arm" was absent from the tRNA-like structure in the 3' untranslated regions (UTRs) of MYFV RNAs. As unique characteristics, MYFV RNA3 lacked the poly(A) tract in the intercistronic region and contained a directly repeated sequence of about 200 nucleotides and polypyrimidine tracts of heterogeneous lengths in the 5' UTR. Co-infection experiments using RNA3 clones with or without the duplicated sequence demonstrated that the duplication contributed to the competitive fitness of the virus in Nicotiana benthamiana.

Host-dependent roles of the viral 5' untranslated region (UTR) in RNA stabilization and cap-independent translational enhancement mediated by the 3' UTR of Red clover necrotic mosaic virus RNA1.

The genome of Red clover necrotic mosaic virus (RCNMV) consists of RNA1 and RNA2, both lacking a cap structure and a poly(A)tail. RNA1 has a translational enhancer element (3'TE-DR1) in the 3' untranslated region (UTR). In this study, we analyzed the roles of 5' and 3' UTRs of RNA1 in 3'TE-DR1-mediated cap-independent translation in cowpea and tobacco BY-2 protoplasts using a dual-luciferase (Luc) reporter assay system. Most mutations introduced into RNA1 5' UTR in reporter Luc mRNA abolished or greatly reduced cap-independent translation in BY-2 protoplasts, whereas those mutations had no or much milder effects if any on translational activity in cowpea protoplasts. Our results suggest that a stem-loop structure predicted in the 5' proximal region of RNA1 plays important roles in both translation and RNA stability. We also show that 3'TE-DR1-mediated cap-independent translation relies on a ribosome-scanning mechanism in both protoplasts.

A viral noncoding RNA generated by cis-element-mediated protection against 5'->3' RNA decay represses both cap-independent and cap-dependent translation.

Positive-strand RNA viruses use diverse mechanisms to regulate viral and host gene expression for ensuring their efficient proliferation or persistence in the host. We found that a small viral noncoding RNA (0.4 kb), named SR1f, accumulated in Red clover necrotic mosaic virus (RCNMV)-infected plants and protoplasts and was packaged into virions. The genome of RCNMV consists of two positive-strand RNAs, RNA1 and RNA2. SR1f was generated from the 3' untranslated region (UTR) of RNA1, which contains RNA elements essential for both cap-independent translation and negative-strand RNA synthesis. A 58-nucleotide sequence in the 3' UTR of RNA1 (Seq1f58) was necessary and sufficient for the generation of SR1f. SR1f was neither a subgenomic RNA nor a defective RNA replicon but a stable degradation product generated by Seq1f58-mediated protection against 5'-->3' decay. SR1f efficiently suppressed both cap-independent and cap-dependent translation both in vitro and in vivo. SR1f trans inhibited negative-strand RNA synthesis of RCNMV genomic RNAs via repression of replicase protein production but not via competition of replicase proteins in vitro. RCNMV seems to use cellular enzymes to generate SR1f that might play a regulatory role in RCNMV infection. Our results also suggest that Seq1f58 is an RNA element that protects the 3'-side RNA sequences against 5'-->3' decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae.

cis-Acting core RNA elements required for negative-strand RNA synthesis and cap-independent translation are separated in the 3'-untranslated region of Red clover necrotic mosaic virus RNA1.

The genome of Red clover necrotic mosaic virus (RCNMV) is positive-sense and divided into RNA1 and RNA2. RNA1 has a translation enhancer element (3' TE-DR1) in the 3' untranslated region (UTR) that substitutes for a 5' cap. In this study, we determined the regions required for cap-independent translation and RNA synthesis in the 3' UTR of RNA1 using a cell-free extract of evacuolated BY-2 protoplasts (BYL) and by an assay in BY-2 protoplasts. The use of capped viral RNA transcripts in the BYL system allowed us to distinguish the effects of introduced mutations on cap-independent translation and negative-strand RNA synthesis of RNA1. We found that the core RNA element of 3' TE-DR1 essential for cap-independent translation of RNA1 is dispensable for negative-strand RNA synthesis. Thus, cis-acting RNA elements essential for cap-independent translation are separated from those required for negative-strand RNA synthesis in the 3' UTR of RCNMV RNA1.

Downregulation of the NbNACa1 gene encoding a movement-protein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana.

The 3a movement protein (MP) plays a central role in the movement of the RNA plant virus, Brome mosaic virus (BMV). To identify host factor genes involved in viral movement, a cDNA library of Nicotiana benthamiana, a systemic host for BMV, was screened with far-Western blotting using a recombinant BMV MP as probe. One positive clone encoded a protein with sequence similarity to the alpha chain of nascent-polypeptide-associated complex from various organisms, which is proposed to contribute to the fidelity of translocation of newly synthesized proteins. The orthologous gene from N. benthamiana was designated NbNACa1. The binding of NbNACa1 to BMV MP was confirmed in vivo with an agroinfiltration-immunoprecipitation assay. To investigate the involvement of NbNACa1 in BMV multiplication, NbNACa1-silenced (GSNAC) transgenic N. benthamiana plants were produced. Downregulation of NbNACa1 expression reduced virus accumulation in inoculated leaves but not in protoplasts. A microprojectile bombardment assay to monitor BMV-MP-assisted viral movement demonstrated reduced virus spread in GSNAC plants. The localization to the cell wall of BMV MP fused to green fluorescent protein was delayed in GSNAC plants. From these results, we propose that NbNACa1 is involved in BMV cell-to-cell movement through the regulation of BMV MP localization to the plasmodesmata.

Cap-independent translation mechanism of red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication.

The genome of Red clover necrotic mosaic virus (RCNMV) in the genus Dianthovirus is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5' end and no poly(A) tail at the 3' end. The 3' untranslated region (3' UTR) of RCNMV RNA1 contains an essential RNA element (3'TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect cis-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3'TE-DR1 of RNA1. However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5' UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in cis-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.

Use of Spring beauty latent virus to identify compatible interactions between bromovirus components required for virus infection.

Spring beauty latent virus (SBLV) is a member of the genus Bromovirus, and is closely related to Brome mosaic virus (BMV) and Cowpea chlorotic mottle virus (CCMV). Compatible interactions between viral components are required for successful infection of plants by BMV and CCMV. To further our understanding of interactions between bromovirus components, we used SBLV to produce reassortants among the three bromoviruses. We found that SBLV RNA 2 functioned with heterologous bromovirus RNA 1 in infections of whole plants and protoplasts of Nicotiana benthamiana, although SBLV RNA 1 did not function with heterologous bromovirus RNA 2. A DNA-based transient assay for 1a and 2a proteins, which are encoded by RNAs 1 and 2, respectively further suggested that SBLV 2a protein may function in combination with heterologous bromovirus 1a protein. Moreover, analysis of the ability of reassortants to spread locally revealed that an RNA 2-mediated interaction between viral components may be required for efficient cell-to-cell movement of bromoviruses.

Identification of a novel RNA silencing suppressor, NSs protein of Tomato spotted wilt virus.

RNA silencing or post-transcriptional gene silencing (PTGS) in plants is known as a defense system against virus infection. Several plant viruses have been shown to encode an RNA silencing suppressor. Using a green fluorescent protein-based transient suppression assay, we show that NSs protein of Tomato spotted wilt virus (TSWV) has RNA silencing suppressor activity. TSWV NSs protein suppressed sense transgene-induced PTGS but did not suppress inverted repeat transgene-induced PTGS. TSWV NSs protein is the first RNA silencing suppressor identified in negative-strand RNA viruses.