Chung-Chi Hu

Satellite RNAs and Satellite Viruses of Plants

The view that satellite RNAs (satRNAs) and satellite viruses are purely molecular parasites of their cognate helper viruses has changed. The molecular mechanisms underlying the synergistic and/or antagonistic interactions among satRNAs/satellite viruses, helper viruses, and host plants are beginning to be comprehended. This review aims to summarize the recent achievements in basic and practical research, with special emphasis on the involvement of RNA silencing mechanisms in the pathogenicity, population dynamics, and, possibly, the origin(s) of these subviral agents. With further research following current trends, the comprehensive understanding of satRNAs and satellite viruses could lead to new insights into the trilateral interactions among host plants, viruses, and satellites.

Identification of differentially expressed genes induced by Bamboo mosaic virus infection in Nicotiana benthamiana by cDNA-amplified fragment length polymorphism

BackgroundThe genes of plants can be up- or down-regulated during viral infection to influence the replication of viruses. Identification of these differentially expressed genes could shed light on the defense systems employed by plants and the mechanisms involved in the adaption of viruses to plant cells. Differential gene expression in Nicotiana benthamiana plants in response to infection with Bamboo mosaic virus (BaMV) was revealed using cDNA-amplified fragment length polymorphism (AFLP).ResultsFollowing inoculation with BaMV, N. benthamiana displayed differential gene expression in response to the infection. Isolation, cloning, and sequencing analysis using cDNA-AFLP furnished 90 cDNA fragments with eight pairs of selective primers. Fifteen randomly selected genes were used for a combined virus-induced gene silencing (VIGS) knockdown experiment, using BaMV infection to investigate the roles played by these genes during viral infection, specifically addressing the means by which these genes influence the accumulation of BaMV protein. Nine of the 15 genes showed either a positive or a negative influence on the accumulation of BaMV protein. Six knockdown plants showed an increase in the accumulation of BaMV, suggesting that they played a role in the resistance to viral infection, while three plants showed a reduction in coat protein, indicating a positive influence on the accumulation of BaMV in plants. An interesting observation was that eight of the nine plants showing an increase in BaMV coat protein were associated with cell rescue, defense, death, aging, signal transduction, and energy production.ConclusionsThis study reports an efficient and straightforward method for the identification of host genes involved in viral infection. We succeeded in establishing a cDNA-AFLP system to help track changes in gene expression patterns in N. benthamiana plants when infected with BaMV. The combination of both DNA-AFLP and VIGS methodologies made it possible to screen a large number of genes and identify those associated with infections of plant viruses. In this report, 9 of the 15 analyzed genes exhibited either a positive or a negative influence on the accumulation of BaMV in N. benthamiana plants.

Transgenic resistance to Cymbidium mosaic virus in Dendrobium expressing the viral capsid protein gene.

A Taiwan isolate of Cymbidium mosaic virus (CymMV-CS) was isolated from infected Cymbidium sinesis Willd. The cDNA of the capsid protein (CP) gene was synthesized and sequenced. Alignment of this CP gene with other reported CPs revealed homologies of 92–98% at the nucleotide level and 98–99% at the amino acid level. To generate virus-resistant varieties, the CymMV-CS CP gene was transformed into Dendrobium protocorms through particle bombardment. Transformants were selected on medium supplemented with 20 mg/L hygromycin and the presence of the transgene was confirmed by polymerase chain reaction, Southern, Northern and Western blot analyses. Transgenic Dendrobium harboring the CymMV CP gene expressed a very low level of virus accumulation four months post-inoculation with CymMV, as detected by ELISA. The transgenic plants exhibited much milder symptoms than the non-transgenic plants upon challenge with CymMV virions

Induction of protective immunity in chickens immunized with plant-made chimeric Bamboo mosaic virus particles expressing very virulent Infectious bursal disease virus antigen

Very virulent Infectious bursal disease virus (vvIBDV) causes a highly contagious disease in young chickens and leads to significant economic loss in the poultry industry. Effective new vaccines are urgently needed. Autonomously replicating plant virus-based vector provides attractive means for producing chimeric virus particles (CVPs) in plants that can be developed into vaccines. In this study, we demonstrate the potential for vaccine development of Bamboo mosaic virus (BaMV) epitope-presentation system, where the antigen from vvIBDV VP2 was fused to the N-terminus of BaMV coat protein. Accordingly, an infections plasmid, pBIBD2, was constructed. Inoculation of the recombinant BaMV clone pBIBD2 enabled the generation of chimeric virus, BIBD2, and stable expression of IBDV VP2 antigen on its coat protein. After intramuscular immunization with BIBD2 CVPs, chickens produced antibodies against IBDV and were protected from vvIBDV (V263/TW strain) challenges. These results corroborate the feasibility of BaMV-based CVP platform in plants for the development and production of vaccines against IBDV.

Unusual roles of host metabolic enzymes and housekeeping proteins in plant virus replication

Viruses have developed the ability to improvise their own replication machineries with host proteins, adapt to different environments, and overcome difficulties encountered during various stages of their infection cycles. The modular nature of protein functional motifs allows for the novel use of ordinary host factors. Recent studies have revealed that positive-sense RNA [(+)RNA] viruses may adapt regular metabolic enzymes and housekeeping proteins of host plants by exploiting unusual functions to accommodate their need for replication, mainly for recruitment and subcellular localization of RNA templates or components of replicase complexes and for controlling switches in different stages of replication. This review compares the newly discovered roles of selected metabolic enzymes and housekeeping proteins in plant (+)RNA virus replication with their original cellular functions and the different consequences when utilized by different viruses.

The two conserved cysteine residues of the triple gene block protein 2 are critical for both cell-to-cell and systemic movement of Bamboo mosaic virus.

The triple gene block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) is a transmembrane protein which is known to be required for the cell-to-cell movement of potexviruses. This protein has two conserved Cys residues, Cys-109 and Cys-112, at its C-terminal tail, which is supposed to be exposed on the outer surface of the endoplasmic reticulum (ER) membrane and ER-derived granular vesicles. In this study, we investigated the importance of these two Cys residues on the cell-to-cell and systemic movement of BaMV. Our results indicate that the Cys-to-Ala substitutions in TGBp2 make the cell-to-cell movement of BaMV relatively inefficient and the systemic movement of BaMV severely inhibited. Moreover, the defect in systemic movement is attributed to the inefficient transport of viral RNA in the phloem of petiole. Clearly, TGBp2 is critical not only for the cell-to-cell but also for the systemic movement of BaMV. In addition, the conserved Cys residues are important for the functioning of TGBp2.

Phosphorylation of Coat Protein by Protein Kinase CK2 Regulates Cell-to-Cell Movement of Bamboo mosaic virus Through Modulating RNA Binding

In this study, we investigated the fine regulation of cell-to-cell movement of Bamboo mosaic virus (BaMV). We report that the coat protein (CP) of BaMV is phosphorylated in planta at position serine 241 (S241), in a process involving Nicotiana benthamiana casein kinase 2α (NbCK2α). BaMV CP and NbCK2α colocalize at the plasmodesmata, suggesting that phosphorylation of BaMV may be involved in its movement. S241 was mutated to examine the effects of temporal and spatial dysregulation of phosphorylation on i) the interactions between CP and viral RNA and ii) the regulation of cell-to-cell movement. Replacement of S241 with alanine did not affect RNA binding affinity but moderately impaired cell-to-cell movement. A negative charge at position 241 reduced the ability of CP to bind RNA and severely interfered with cell-to-cell movement. Deletion of residues 240 to 242 increased the affinity of CP to viral RNA and dramatically impaired cell-to-cell movement. A threonine at position 241 changed the binding preference of CP toward genomic RNA and inhibited cell-to-cell movement. Together, these results reveal a fine regulatory mechanism for the cell-to-cell movement of BaMV, which involves the modulation of RNA binding affinity through appropriate phosphorylation of CP by NbCK2α.

Mimicry of molecular pretenders: the terminal structures of satellites associated with plant RNA viruses.

Satellite RNAs (satRNAs) and satellite viruses depend on the replicase complexes provided by their cognate helper viruses and host plants for replication, pretending that they are part of the viral genomes. Although satRNAs and satellite viruses do not share significant nucleotide sequence similarity with the helper viruses, the essential cis-acting elements recognized by the replicase complexes must reside on their genomes, acting as the mimicry for the molecular pretenders. By understanding how this molecular mimicry deceives the helper viruses into supporting the satellites, a significant amount of knowledge of the basic requirements and mechanisms for replication of viruses and satellites has been obtained. Here we review the recent advances in understanding the effects of the cis elements at the termini of satRNAs and satellite viruses on their accumulation. Several well-characterized satellite/helper virus systems, representing the non-coding short satRNAs, mRNA-type long satRNAs, circular satRNAs, and satellite viruses, are compared and contrasted. It is concluded that different satellites may adopt different strategies to exploit the replication/transcription/translation machineries of their helper viruses, and different mimicries may be implemented by the same molecular pretender for different biological functions.

Viral elements and host cellular proteins in intercellular movement of Bamboo mosaic virus

As a member of the genus Potexvirus, Bamboo mosaic virus (BaMV) also belongs to the plant viruses that encode triple gene block proteins (TGBps) for intercellular movement within the host plants. Recent studies of the movement mechanisms of BaMV have revealed similarities and differences between BaMV and other potexviruses. This review focuses on the general aspects of viral and host elements involved in BaMV movement, the interactions among these elements, and the possible pathways for intra- and intercellular trafficking of BaMV. Major features of BaMV trafficking that have not been demonstrated in other potexviruses include: (i) the involvement of replicase, (ii) fine regulation by coat protein phosphorylation, (iii) the key roles played by TGBp3, (iv) the use of virions as the major transported form, and (v) the involvement of specific host factors, such as Ser/Thr kinase-like protein of Nicotiana benthamiana. We also highlight areas for future study that will provide a more comprehensive understanding of the detailed interactions among viral movement proteins and host factors, as well as the regulatory mechanisms of virus movement. Finally, a model based on the current knowledge is proposed to depict the diverse abilities of BaMV to utilize a wide range of mechanisms for efficient intercellular movement.

Production of Japanese Encephalitis Virus Antigens in Plants Using Bamboo Mosaic Virus-Based Vector

Japanese encephalitis virus (JEV) is among the major threats to public health in Asia. For disease control and prevention, the efficient production of safe and effective vaccines against JEV is in urgent need. In this study, we produced a plant-made JEV vaccine candidate using a chimeric virus particle (CVP) strategy based on bamboo mosaic virus (BaMV) for epitope presentation. The chimeric virus, designated BJ2A, was constructed by fusing JEV envelope protein domain III (EDIII) at the N-terminus of BaMV coat protein, with an insertion of the foot-and-mouth disease virus 2A peptide to facilitate the production of both unfused and epitope-presenting for efficient assembly of the CVP vaccine candidate. The strategy allowed stable maintenance of the fusion construct over long-term serial passages in plants. Immuno-electron microscopy examination and immunization assays revealed that BJ2A is able to present the EDIII epitope on the surface of the CVPs, which stimulated effective neutralizing antibodies against JEV infection in mice. This study demonstrates the efficient production of an effective CVP vaccine candidate against JEV in plants by the BaMV-based epitope presentation system.