Publications by authors named "Kiwamu Hyodo"

24 Publications

  • Page 1 of 1

Identification of an RNA Silencing Suppressor Encoded by a Symptomless Fungal Hypovirus, Cryphonectria Hypovirus 4.

Biology (Basel) 2021 Jan 31;10(2). Epub 2021 Jan 31.

Institute of Plant Science and Resources (IPSR), Okayama University, Kurashiki 710-0046, Japan.

Previously, we have reported the ability of a symptomless hypovirus Cryphonectria hypovirus 4 (CHV4) of the chestnut blight fungus to facilitate stable infection by a co-infecting mycoreovirus 2 (MyRV2)-likely through the inhibitory effect of CHV4 on RNA silencing (Aulia et al., Virology, 2019). In this study, the N-terminal portion of the CHV4 polyprotein, termed p24, is identified as an autocatalytic protease capable of suppressing host antiviral RNA silencing. Using a bacterial expression system, CHV4 p24 is shown to cleave autocatalytically at the di-glycine peptide (Gly214-Gly215) of the polyprotein through its protease activity. Transgenic expression of CHV4 p24 in suppresses the induction of one of the key genes of the antiviral RNA silencing, dicer-like 2, and stabilizes the infection of RNA silencing-susceptible virus MyRV2. This study shows functional similarity between CHV4 p24 and its homolog p29, encoded by the symptomatic prototype hypovirus CHV1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biology10020100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912522PMC
January 2021

Hijacking of host cellular components as proviral factors by plant-infecting viruses.

Adv Virus Res 2020 25;107:37-86. Epub 2020 May 25.

Department of Plant Life Science, Faculty of Agriculture, Ryukoku University, Otsu, Shiga, Japan.

Plant viruses are important pathogens that cause serious crop losses worldwide. They are obligate intracellular parasites that commandeer a wide array of proteins, as well as metabolic resources, from infected host cells. In the past two decades, our knowledge of plant-virus interactions at the molecular level has exploded, which provides insights into how plant-infecting viruses co-opt host cellular machineries to accomplish their infection. Here, we review recent advances in our understanding of how plant viruses divert cellular components from their original roles to proviral functions. One emerging theme is that plant viruses have versatile strategies that integrate a host factor that is normally engaged in plant defense against invading pathogens into a viral protein complex that facilitates viral infection. We also highlight viral manipulation of cellular key regulatory systems for successful virus infection: posttranslational protein modifications for fine control of viral and cellular protein dynamics; glycolysis and fermentation pathways to usurp host resources, and ion homeostasis to create a cellular environment that is beneficial for viral genome replication. A deeper understanding of viral-infection strategies will pave the way for the development of novel antiviral strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.aivir.2020.04.002DOI Listing
April 2021

Diverse Partitiviruses From the Phytopathogenic Fungus, .

Front Microbiol 2020 26;11:1064. Epub 2020 Jun 26.

Institute of Plant Science and Resources, Okayama University, Kurashiki, Japan.

Partitiviruses (dsRNA viruses, family ) are ubiquitously detected in plants and fungi. Although previous surveys suggested their omnipresence in the white root rot fungus, , only a few of them have been molecularly and biologically characterized thus far. We report the characterization of a total of 20 partitiviruses from 16 strains belonging to 15 new species, for which "-" were proposed, and 5 previously reported species. The newly identified partitiviruses have been taxonomically placed in two genera, , and . Some partitiviruses were transfected into reference strains of the natural host, , and an experimental host, , using purified virions. A comparative analysis of resultant transfectants revealed interesting differences and similarities between the RNA accumulation and symptom induction patterns of and . Other interesting findings include the identification of a probable reassortment event and a quintuple partitivirus infection of a single fungal strain. These combined results provide a foundation for further studies aimed at elucidating mechanisms that underly the differences observed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2020.01064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332551PMC
June 2020

Virome Analysis of Aphid Populations That Infest the Barley Field: The Discovery of Two Novel Groups of Nege/Kita-Like Viruses and Other Novel RNA Viruses.

Front Microbiol 2020 7;11:509. Epub 2020 Apr 7.

Institute of Plant Science and Resources (IPSR), Okayama University, Kurashiki, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2020.00509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154061PMC
April 2020

Two novel fungal negative-strand RNA viruses related to mymonaviruses and phenuiviruses in the shiitake mushroom (Lentinula edodes).

Virology 2019 07 23;533:125-136. Epub 2019 May 23.

Institute of Plant Science and Resources (IPSR), Okayama University, Kurashiki 710-0046, Japan. Electronic address:

There is still limited information on the diversity of (-)ssRNA viruses that infect fungi. Here, we have discovered two novel (-)ssRNA mycoviruses in the shiitake mushroom (Lentinula edodes). The first virus has a monopartite RNA genome and relates to that of mymonaviruses (Mononegavirales), especially to Hubei rhabdo-like virus 4 from arthropods and thus designated as Lentinula edodes negative-strand RNA virus 1. The second virus has a putative bipartite RNA genome and is related to the recently discovered bipartite or tripartite phenui-like viruses (Bunyavirales) associated with plants and ticks, and designated as Lentinula edodes negative-strand RNA virus 2 (LeNSRV2). LeNSRV2 is likely the first segmented (-)ssRNA virus known to infect fungi. Its smaller RNA segment encodes a putative nucleocapsid and a plant MP-like protein using a potential ambisense coding strategy. These findings enhance our understanding of the diversity, evolution and spread of (-)ssRNA viruses in fungi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2019.05.008DOI Listing
July 2019

Hijacking a host scaffold protein, RACK1, for replication of a plant RNA virus.

New Phytol 2019 01 31;221(2):935-945. Epub 2018 Aug 31.

Department of Plant Life Science, Faculty of Agriculture, Ryukoku University, Otsu, Shiga, 520-2194, Japan.

Receptor for activated C kinase 1 (RACK1) is strictly conserved across eukaryotes and acts as a versatile scaffold protein involved in various signaling pathways. Plant RACK1 is known to exert important functions in innate immunity against fungal and bacterial pathogens. However, the role of the RACK1 in plant-virus interactions remains unknown. Here, we addressed the role of RACK1 of Nicotiana benthamiana during infection by red clover necrotic mosaic virus (RCNMV), a plant positive-stranded RNA virus. NbRACK1 was shown to be recruited by the p27 viral replication protein into endoplasmic reticulum-derived aggregated structures (possible replication sites). Downregulation of NbRACK1 by virus-induced gene silencing inhibited viral cap-independent translation and p27-mediated reactive oxygen species (ROS) accumulation, which are prerequisite for RCNMV replication. We also found that NbRACK1 interacted with a host calcium-dependent protein kinase (NbCDPKiso2) that activated a ROS-generating enzyme. Interestingly, NbRACK1 was required for the interaction of p27 with NbCDPKiso2, suggesting that NbRACK1 acts as a bridge between the p27 viral replication protein and NbCDPKiso2. Collectively, our findings provide an example of a viral strategy in which a host multifaceted scaffold protein RACK1 is highjacked for promoting viral protein-triggered ROS production necessary for robust viral replication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/nph.15412DOI Listing
January 2019

The Photosystem II Repair Cycle Requires FtsH Turnover through the EngA GTPase.

Plant Physiol 2018 10 21;178(2):596-611. Epub 2018 Aug 21.

Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama 710-0046, Japan

Specific degradation of photodamaged D1, the photosystem II (PSII) reaction center protein, is a crucial step in the PSII repair cycle to maintain photosynthesis activity. Processive proteolysis by the FtsH protease is fundamental to cooperative D1 degradation. Here, we attempted to purify the FtsH complex to elucidate its regulation mechanisms and substrate recognition in Arabidopsis (). Unlike previously reported prokaryotic and mitochondrial FtsHs, the Arabidopsis chloroplastic FtsH does not appear to form a megacomplex with prohibition-like proteins but instead accumulates as smaller complexes. The copurified fraction was enriched with a partial PSII intermediate presumably undergoing repair, although its precise properties were not fully clarified. In addition, we copurified a bacteria-type GTPase localized in chloroplasts, EngA, and confirmed its interaction with FtsH by subsequent pull-down and bimolecular fluorescence complementation assays. While the mutation is embryo lethal, the transgenic lines overexpressing EngA (EngA-OX) showed leaf variegation reminiscent of the variegated mutant lacking FtsH2. EngA-OX was revealed to accumulate more cleaved D1 fragments and reactive oxygen species than the wild type, indicative of compromised PSII repair. Based on these results and the fact that FtsH becomes more stable in EngA-OX, we propose that EngA negatively regulates FtsH stability. We demonstrate that proper FtsH turnover is crucial for PSII repair in the chloroplasts of Arabidopsis. Consistent with the increased turnover of FtsH under high-light conditions in , our findings underline the rapid turnover of not only D1 but also FtsH proteases in the PSII repair cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1104/pp.18.00652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181060PMC
October 2018

Integration of danger peptide signals with herbivore-associated molecular pattern signaling amplifies anti-herbivore defense responses in rice.

Plant J 2018 05 1;94(4):626-637. Epub 2018 Apr 1.

Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama, 710-0046, Japan.

Plant defense against herbivores is modulated by herbivore-associated molecular patterns (HAMPs) from oral secretions (OS) and/or saliva of insects. Furthermore, feeding wounds initiate plant self-damage responses modulated by danger-associated molecular patterns (DAMPs) such as immune defense-promoting plant elicitor peptides (Peps). While temporal and spatial co-existence of both patterns during herbivory implies a possibility of their close interaction, the molecular mechanisms remain undetermined. Here we report that exogenous application of rice (Oryza sativa) peptides (OsPeps) can elicit multiple defense responses in rice cell cultures. Specific activation of OsPROPEP3 gene transcripts in rice leaves by wounding and OS treatments further suggests a possible involvement of the OsPep3 peptide in rice-herbivore interactions. Correspondingly, we found that simultaneous application of OsPep3 and Mythimna loreyi OS significantly amplifies an array of defense responses in rice cells, including mitogen-activated protein kinase activation, and generation of defense-related hormones and metabolites. The induction of OsPROPEP3/4 by OsPep3 points to a positive auto-feedback loop in OsPep signaling which may contribute to additional enhancement of defense signal(s). Finally, the overexpression of the OsPep receptor OsPEPR1 increases the sensitivity of rice plants not only to the cognate OsPeps but also to OS signals. Our findings collectively suggest that HAMP-DAMP signal integration provides a critical step in the amplification of defense signaling in plants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tpj.13883DOI Listing
May 2018

Chloroplast Genome Sequences of Seven Strains of the Bloom-Forming Raphidophyte .

Genome Announc 2017 Oct 12;5(41). Epub 2017 Oct 12.

Institute of Plant Science and Resources, Okayama University, Okayama, Japan

We report here the complete chloroplast genome sequences of seven strains of the bloom-forming raphidophyte These ~160-kb sequences contain 124 protein-, 6 rRNA-, and 34 tRNA-coding sequences. Notable sequence variations were observed among these seven sequenced and two previously characterized strains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/genomeA.01030-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5637492PMC
October 2017

Dual function of a cis-acting RNA element that acts as a replication enhancer and a translation repressor in a plant positive-stranded RNA virus.

Virology 2017 12 21;512:74-82. Epub 2017 Sep 21.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan; Department of Plant Life Science, Faculty of Agriculture, Ryukoku University, Otsu, Shiga 520-2194, Japan. Electronic address:

The genome of red clover necrotic mosaic virus is divided into two positive-stranded RNA molecules of RNA1 and RNA2, which have no 5' cap structure and no 3' poly(A) tail. Previously, we showed that any mutations in the cis-acting RNA replication elements of RNA2 abolished its cap-independent translational activity, suggesting a strong link between RNA replication and translation. Here, we investigated the functions of the 5' untranslated region (UTR) of RNA2 and revealed that the basal stem-structure (5'BS) predicted in the 5' UTR is essential for robust RNA replication. Interestingly, RNA2 mutants with substitution or deletion in the right side of the 5'BS showed strong translational activity, despite their impaired replication competency. Furthermore, nucleotide sequences other than the 5'BS of the 5' UTR were essential to facilitate the replication-associated translation. Overall, these cis-acting RNA elements seem to coordinately regulate the balance between RNA replication and replication-associated translation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2017.09.008DOI Listing
December 2017

Requirement for eukaryotic translation initiation factors in cap-independent translation differs between bipartite genomic RNAs of red clover necrotic mosaic virus.

Virology 2017 09 21;509:152-158. Epub 2017 Jun 21.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan; Department of Plant Life Science, Faculty of Agriculture, Ryukoku University, Otsu, Shiga 520-2194, Japan. Electronic address:

The bipartite genomic RNAs of red clover necrotic mosaic virus (RCNMV) lack a 5' cap and a 3' poly(A) tail. RNA1 encodes viral replication proteins, and RNA2 encodes a movement protein (MP). These proteins are translated in a cap-independent manner. We previously identified two cis-acting RNA elements that cooperatively recruit eukaryotic translation initiation factor (eIF) complex eIF4F or eIFiso4F to RNA1. Such cis-acting RNA elements and host factors have not been identified in RNA2. Here we found that translation of RNA1 was significantly compromised in Arabidopsis thaliana carrying eif4f mutation. RNA1 replicated efficiently in eifiso4f1 mutants, suggesting vigorous translation of the replication proteins from RNA1 in the plants. In contrast, MP accumulation was decreased in eifiso4f1 mutants but not in eif4f mutants. Collectively, these results suggest that RCNMV uses different eIF complexes for translation of its bipartite genomic RNAs, which may contribute to fine-tuning viral gene expression during infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2017.06.015DOI Listing
September 2017

Roles of superoxide anion and hydrogen peroxide during replication of two unrelated plant RNA viruses in Nicotiana benthamiana.

Plant Signal Behav 2017 06 8;12(6):e1338223. Epub 2017 Jun 8.

b Laboratory of Plant Pathology , Graduate School of Agriculture, Kyoto University , Sakyo-ku , Kyoto , Japan.

Reactive oxygen species (ROS), including superoxide anion (O), hydrogen peroxide (HO), and hydroxyl radical, act as signaling molecules to transduce biotic and abiotic stimuli into stress adaptations in plants. A respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) is responsible for O production to inhibit pathogen infection during plant innate immunity. RBOH-derived O can be immediately converted into HO by the action of superoxide dismutase. Interestingly, we recently showed that red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, hijacks the host's ROS-generating machinery during infection. An RCNMV replication protein associates with NbRBOHB and triggers intracellular ROS bursts. These bursts are required for robust viral RNA replication. However, what types of ROS are required for viral replication is currently unknown. Here, we found that RCNMV replication was sensitive to an O scavenger but insensitive to an HO scavenger. Interestingly, replication of another plant (+)RNA virus, brome mosaic virus, was sensitive to both types of scavengers. These results indicate a virus-specific pattern requirement of O and HO for (+)RNA virus replication and suggest a conserved nature of the roles of ROS in (+)RNA virus replication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15592324.2017.1338223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566351PMC
June 2017

Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus.

Proc Natl Acad Sci U S A 2017 02 1;114(7):E1282-E1290. Epub 2017 Feb 1.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan;

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 (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 (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 RBOH-derived 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1610212114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320965PMC
February 2017

Pathogenesis mediated by proviral host factors involved in translation and replication of plant positive-strand RNA viruses.

Curr Opin Virol 2016 04 30;17:11-18. Epub 2015 Nov 30.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Electronic address:

Viral pathogenesis comes from complex interactions between viruses and hosts. All the processes of viral infection, including translation of viral factors and replication of viral genomes, define viral pathogenesis; therefore, molecular insights into the mechanisms underlying viral replication strategies unambiguously pave the way for our comprehensive understanding of viral pathogenesis and disease outcome, as well as for developing new antiviral strategies against plant virus disease. Recent studies of plant positive-strand RNA [(+)RNA] viruses have advanced our understanding of co-opted host factors and their roles in viral translation and replication. It is becoming clear that plant (+)RNA viruses harness host factors involved in membrane trafficking and lipid metabolism to establish the viral replication complex (VRC). In this review, we aim to discuss the contribution of co-opted host factors in translation and genome replication of plant (+)RNA viruses mainly focusing on those involved in the biogenesis of the VRC, which may act as a central hub in almost all the processes of viral infection as well as viral pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.coviro.2015.11.004DOI Listing
April 2016

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

PLoS Pathog 2015 May 28;11(5):e1004909. Epub 2015 May 28.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1004909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447390PMC
May 2015

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

PLoS Pathog 2014 Nov 20;10(11):e1004505. Epub 2014 Nov 20.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1004505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239097PMC
November 2014

Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes.

Front Plant Sci 2014 7;5:321. Epub 2014 Jul 7.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2014.00321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083346PMC
July 2014

Traffic jam on the cellular secretory pathway generated by a replication protein from a plant RNA virus.

Plant Signal Behav 2014 8;9(3):e28644. Epub 2014 Apr 8.

Graduate School of Agriculture; Kyoto University; Kyoto, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091560PMC
http://dx.doi.org/10.4161/psb.28644DOI Listing
April 2015

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

J Virol 2013 Jan 24;87(1):163-76. Epub 2012 Oct 24.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02383-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536388PMC
January 2013

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

J Virol 2012 Nov 29;86(22):12091-104. Epub 2012 Aug 29.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01659-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486462PMC
November 2012

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

Virology 2012 Nov 14;433(1):131-41. Epub 2012 Aug 14.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2012.07.017DOI Listing
November 2012

Identification of amino acids in auxiliary replicase protein p27 critical for its RNA-binding activity and the assembly of the replicase complex in Red clover necrotic mosaic virus.

Virology 2011 May 26;413(2):300-9. Epub 2011 Mar 26.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606–8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2011.02.017DOI Listing
May 2011

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

J Virol 2011 Jan 27;85(1):497-509. Epub 2010 Oct 27.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01754-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3014169PMC
January 2011

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.

Virology 2010 Nov 15;407(2):213-24. Epub 2010 Sep 15.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2010.07.038DOI Listing
November 2010