Molecular biology and epidemiology of dianthoviruses.

The genus Dianthovirus is one of eight genera in the family Tombusviridae. All the genera have monopartite positive-stranded RNA genomes, except the dianthoviruses which have bipartite genomes. The dianthoviruses are distributed worldwide. Although they share common structural features with the other Tombusviridae viruses in their virions and the terminal structure of the genomic RNAs, the bipartite nature of the dianthovirus genome offers an ideal experimental system with which to study basic issues of virology. The two genomic RNAs seem to use distinct strategies to regulate their translation, transcription, genome replication, genome packaging, and cell-to-cell movement during infection. This review summarizes the current state of our knowledge of the dianthoviruses, with its main emphasis on the molecular biology of the virus, including the viral and host factors required for its infection of host plants. The epidemiology of the virus and the possible viral impacts on agriculture and the environment are also discussed

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

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, 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

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

A Modeling of Singing Voice Robust to Accompaniment Sounds and Its Application to Singer Identification and Vocal-Timbre-Similarity-Based Music Information Retrieval

This paper describes a method of modeling the characteristics of a singing voice from polyphonic musical audio signals including sounds of various musical instruments. Because singing voices play an important role in musical pieces with vocals, such representation is useful for music information retrieval systems. The main problem in modeling the characteristics of a singing voice is the negative influences caused by accompaniment sounds. To solve this problem, we developed two methods, accompaniment sound reduction and reliable frame selection . The former makes it possible to calculate feature vectors that represent a spectral envelope of a singing voice after reducing accompaniment sounds. It first extracts the harmonic components of the predominant melody from sound mixtures and then resynthesizes the melody by using a sinusoidal model driven by these components. The latter method then estimates the reliability of frame of the obtained melody (i.e., the influence of accompaniment sound) by using two Gaussian mixture models (GMMs) for vocal and nonvocal frames to select the reliable vocal portions of musical pieces. Finally, each song is represented by its GMM consisting of the reliable frames. This new representation of the singing voice is demonstrated to improve the performance of an automatic singer identification system and to achieve an MIR system based on vocal timbre similarity

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

AbstractRed 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 indentified 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

逆転写酵素・DNAポリメラーゼを用いたRT-PCRによる増幅反応で確認されたペッパーマイルドモットルウイルス（PMMoV）感染ピーマンの果実種子におけるウイルスの効果的な不活性化方法

A method for efficient inactivation Papper mild mottle virus (PMMoV) in harvested seeds of green pepper was examined based on the infectivity on the leaves of Nicotina glutinosa L.(a local host) and a reverse transcription and polymerase chain reaction (RT-PCR)-based amplification of the viral RNA. The seed homogenates from PMMoV-infected plants produced a large number of necrotic local lesion in N.glutinosa, but soaking the seeds in 10% (w/v) tri-sodium phosphate (Na3PO4) for 20 min or dry sterilization (70C,3h) effectivity eliminated the abundance of PMMoV. However, no necrotic lesion on N.glutinosa or the RT-PCR-basaed amplification was observed with seeds that has been disinfected by Na3PO4 in combination with dry sterilization

Dual induction of caspase 3- and transglutaminase-dependent apoptosis by acyclic retinoid in hepatocellular carcinoma cells

<p>Abstract</p> <p>Background</p> <p>Hepatocellular carcinoma has a high mortality rate due to its rate of recurrence. Acyclic retinoid prevents recurrence of hepatocellular carcinoma in patients after surgical removal of their primary tumors by inducing apoptosis in hepatocellular carcinoma cells, although the molecular mechanisms of action are not understood.</p> <p>Methods</p> <p>Human hepatocellular carcinoma cells in culture, as well as nude mice transplanted with hepatocellular carcinoma cells and rats given with <it>N</it>-diethylnitrosamine were treated with acyclic retinoid. Changes in activated caspase 3 and transglutaminase 2 (TG2) levels, Sp1 cross-linking and its activities, expression of epidermal growth factor receptor, and apoptotic levels were measured.</p> <p>Results</p> <p>Acyclic retinoid simultaneously stimulated the activation of caspase 3, and the expression, nuclear localization and crosslinking activity of TG2, resulting in crosslinking and inactivation of the transcription factor, Sp1, thereby reducing expression of epidermal growth factor receptor and cell death in three hepatocellular carcinoma cell lines. These effects were partially restored by a caspase inhibitor, transfection of antisense TG2, restoration of functional Sp1, or an excess of epidermal growth factor. Nuclear expression of TG2 and crosslinked Sp1, as also activated caspase 3 were found in both hepatocellular carcinoma cells transplanted into nude mice and cancerous regions within the liver in <it>N</it>-diethylnitrosamine-induced hepatocarcinogenesis model in rats, following treatment of animals with acyclic retinoid.</p> <p>Conclusions</p> <p>Treatment with acyclic retinoid produces a dual activation of caspase 3 and TG2 induced apoptosis of hepatocellular carcinoma cells via modification and inactivation of Sp1, resulting in reduced expression of epidermal growth factor receptor.</p

Roles of Phosphatidic Acid in Virus RNA Replication

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

ピーマン(Capsicum annuum L.)に導入されたＬ２抵抗性遺伝子を打破する日本産トバモウイルス系統ペッパーマイルドモットルウイルス(PMMoV)の疫学的調査

To understand the epidemiological aspects of tobamovirus infecting the L resistance genotypes of green pepper, fifteen isolates were collected from geographically different fields and were chracterized by their biological properties. All isolates infected L1 and L2 plants systemically, but were localized in L3 and L4 plants. The symptomatology on several test plants and the reactivity to an antiserum showed that they were identical to that of a Japanese strain of pepper mild mottle virus (PMMoV-J). The viral infection was also confirmed by a reverse transcription and polymerase chain reaction (RT-PCR) with oligonucleotide primers that amplity the coat protein gene of PMMoV-RNA. On the other hand, the RT-PCR allowed us to detect PMMoV in seeds of some commercial cultivars of green pepper. Viruses isolated from the seeds could infect L2 plants systemically. Further analysis of the nucleotide sequence of the predicted coat protein gene revealed that the isolates from the commercial seeds were identical to that of PMMoV-J. These results indicated that the L2 resistance-breaking tobamovirus has prevailed in fields of green pepper in Japan. and that infected seeds may be one of the initial sources of the viral infection