Gn protein expressed in plants for diagnosis of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) causes the highly fatal disease in humans. To facilitate diagnosis, the native form of subunit glycoprotein (Gn), a prime target for potential vaccines and therapies, was produced in Nicotiana benthamiana using a Bamboo mosaic virus-based vector system. By fusion with secretory signal tags, SSExt, derived from the extension protein, and the (SP)10 motif, the yield of the recombinant Gn (rGn) was remarkably increased to approximately 7 mg/kg infiltrated leaves. Ultimately, an rGn-based ELISA was successfully established for the detection of SFTSV-specific antibodies in serum samples from naturally infected monkeys. As validated with the reference method, the specificity and sensitivity of rGn-ELISA were 94% and 96%, respectively. In conclusion, utilizing well-suited fusion tags facilitates rGn production and purification in substantial quantities while preserving its antigenic properties. The rGn-ELISA, characterized by its commendable sensitivity and specificity could serve as a viable alternative diagnostic method for assessing SFTSV seroprevalence. KEY POINTS: • SFTSV Gn, fused with secretory signal tags, was expressed by the BaMV-based vector. • The plant fusion tags increased expression levels and eased the purification of rGn. • The rGn-ELISA was established and validated; its specificity and sensitivity > 94%.

Importin α2 participates in RNA interference against bamboo mosaic virus accumulation in Nicotiana benthamiana via NbAGO10a-mediated small RNA clearance.

Karyopherins, the nucleocytoplasmic transporters, participate in multiple RNA silencing stages by transporting associated proteins into the nucleus. Importin α is a member of karyopherins and has been reported to facilitate virus infection via nuclear import of viral proteins. Unlike other RNA viruses, silencing of importin α2 (α2i) by virus-induced gene silencing (VIGS) boosted the titre of bamboo mosaic virus (BaMV) in protoplasts, and inoculated and systemic leaves of Nicotiana benthamiana. The enhanced BaMV accumulation in importin α2i plants was linked to reduced levels of RDR6-dependent secondary virus-derived small-interfering RNAs (vsiRNAs). Small RNA-seq revealed importin α2 silencing did not affect the abundance of siRNAs derived from host mRNAs but significantly reduced the 21 and 22 nucleotide vsiRNAs in BaMV-infected plants. Deletion of BaMV TGBp1, an RNA silencing suppressor, compromised importin α2i-mediated BaMV enhancement. Moreover, silencing of importin α2 upregulated NbAGO10a, a proviral protein recruited by TGBp1 for BaMV vsiRNAs clearance, but hindered the nuclear import of NbAGO10a. Taken together, these results indicate that importin α2 acts as a negative regulator of BaMV invasion by controlling the expression and nucleocytoplasmic shuttling of NbAGO10a, which removes vsiRNAs via the TGBp1-NbAGO10a-SDN1 pathway. Our findings reveal the hidden antiviral mechanism of importin α2 in countering BaMV infection in N. benthamiana.

Trade-off between local replication and long-distance dissemination during experimental evolution of a satellite RNA.

Satellite RNAs (satRNAs) are molecular parasites that depend on their non-homologous helper viruses (HVs) for essential biological functions. While there are multiple molecular and phylogenetic studies on satRNAs, there is no experimental evolution study on how satRNAs may evolve in common infection conditions. In this study, we serially passaged the Bamboo mosaic virus (BaMV) associated-satRNA (satBaMV) under conditions in which satBaMV either coinfects an uninfected host plant, Nicotiana benthamiana, with BaMV or superinfects a transgenic N. benthamiana expressing the full-length BaMV genome. Single-nucleotide polymorphisms (SNPs) of satBaMV populations were analyzed by deep sequencing. Forty-eight SNPs were identified across four different experimental treatments. Most SNPs are treatment-specific, and some are also ephemeral. However, mutations at positions 30, 34, 63, and 82, all located at the 5' untranslated region (UTR), are universal in all treatments. These universal SNPs are configured into several haplotypes and follow different population dynamics. We constructed isogenic satBaMV strains only differing at positions 30 and 82 and conducted competition experiments in protoplasts and host plants. We found that the haplotype that reached high frequency in protoplasts and inoculation leaves also exhibited poor dissemination to systemic leaves and vice versa, thus suggesting an apparent trade-off between local replication and long-distance dissemination. We posit that the trade-off is likely caused by antagonistic pleiotropy at the 5' UTR. Our findings revealed a hitherto under-explored connection between satRNA genome replication and movement within a host plant. The significance of such a connection during satRNA evolution warrants a more thorough investigation.

Argonaute 5-mediated antiviral defense and viral counter-defense in Nicotiana benthamiana.

The argonaute (AGO) family proteins play a crucial role in preventing viral invasions through the plant antiviral RNA silencing pathway, with distinct AGO proteins recruited for specific antiviral mechanisms. Our previous study revealed that Nicotiana benthamiana AGO5 (NbAGO5) expression was significantly upregulated in response to bamboo mosaic virus (BaMV) infection. However, the roles of NbAGO5 in antiviral mechanisms remained to be explored. In this research, we examined the antiviral functions of NbAGO5 in the infections of different viruses. It was found that the accumulation of NbAGO5 was induced not only at the RNA but also at the protein level following the infections of BaMV, potato virus X (PVX), tobacco mosaic virus (TMV), and cucumber mosaic virus (CMV) in N. benthamiana. To explore the antiviral mechanism and regulatory function of NbAGO5, we generated NbAGO5 overexpression (OE-NbAGO5) and knockout (nbago5) transgenic N. benthamiana lines. Our findings reveal that NbAGO5 provides defense against BaMV, PVX, TMV, and a mutant CMV deficient in 2b gene, but not against the wild-type CMV and turnip mosaic virus (TuMV). Through affinity purification and small RNA northern blotting, we demonstrated that NbAGO5 exerts its antiviral function by binding to viral small interfering RNAs (vsiRNAs). Moreover, we observed that CMV 2b and TuMV HC-Pro interact with NbAGO5, triggering its degradation via the 26S proteasome and autophagy pathways, thereby allowing these viruses to overcome NbAGO5-mediated defense. In addition, TuMV HC-Pro provides another line of counter-defense by interfering with vsiRNA binding by NbAGO5. Our study provides further insights into the antiviral RNA interference mechanism and the complex interplay between NbAGO5 and plant viruses.

The Role of Plant Transcription Factors in the Fight against Plant Viruses.

Plants are vulnerable to the challenges of unstable environments and pathogen infections due to their immobility. Among various stress conditions, viral infection is a major threat that causes significant crop loss. In response to viral infection, plants undergo complex molecular and physiological changes, which trigger defense and morphogenic pathways. Transcription factors (TFs), and their interactions with cofactors and cis-regulatory genomic elements, are essential for plant defense mechanisms. The transcriptional regulation by TFs is crucial in establishing plant defense and associated activities during viral infections. Therefore, identifying and characterizing the critical genes involved in the responses of plants against virus stress is essential for the development of transgenic plants that exhibit enhanced tolerance or resistance. This article reviews the current understanding of the transcriptional control of plant defenses, with a special focus on NAC, MYB, WRKY, bZIP, and AP2/ERF TFs. The review provides an update on the latest advances in understanding how plant TFs regulate defense genes expression during viral infection.

Development of an efficient expression system with large cargo capacity for interrogation of gene function in bamboo based on bamboo mosaic virus.

Bamboo is one of the fastest growing plants among monocotyledonous species and is grown extensively in subtropical regions. Although bamboo has high economic value and produces much biomass quickly, gene functional research is hindered by the low efficiency of genetic transformation in this species. We therefore explored the potential of a bamboo mosaic virus (BaMV)-mediated expression system to investigate genotype-phenotype associations. We determined that the sites between the triple gene block proteins (TGBps) and the coat protein (CP) of BaMV are the most efficient insertion sites for the expression of exogenous genes in both monopodial and sympodial bamboo species. Moreover, we validated this system by individually overexpressing the two endogenous genes ACE1 and DEC1, which resulted in the promotion and suppression of internode elongation, respectively. In particular, this system was able to drive the expression of three 2A-linked betalain biosynthesis genes (more than 4 kb in length) to produce betalain, indicating that it has high cargo capacity and may provide the prerequisite basis for the development of a DNA-free bamboo genome editing platform in the future. Since BaMV can infect multiple bamboo species, we anticipate that the system described in this study will greatly contribute to gene function research and further promote the molecular breeding of bamboo.

A viral movement protein co-opts endoplasmic reticulum luminal-binding protein and calreticulin to promote intracellular movement.

Intracellular movement is an important step for the initial spread of virus in plants during infection. This process requires virus-encoded movement proteins (MPs) and their interaction with host factors. Despite the large number of known host factors involved in the movement of different viruses, little is known about host proteins that interact with one of the MPs encoded by potexviruses, the triple-gene-block protein 3 (TGBp3). The main obstacle lies in the relatively low expression level of potexviral TGBp3 in hosts and the weak or transient nature of interactions. Here, we used TurboID-based proximity labeling to identify the network of proteins directly or indirectly interacting with the TGBp3 of a potexvirus, Bamboo mosaic virus (BaMV). Endoplasmic reticulum (ER) luminal-binding protein 4 and calreticulin 3 of Nicotiana benthamiana (NbBiP4 and NbCRT3, respectively) associated with the functional TGBp3-containing BaMV movement complexes, but not the movement-defective mutant, TGBp3M. Fluorescent microscopy revealed that TGBp3 colocalizes with NbBiP4 or NbCRT3 and the complexes move together along ER networks to cell periphery in N. benthamiana. Loss- and gain-of-function experiments revealed that NbBiP4 or NbCRT3 is required for the efficient spread and accumulation of BaMV in infected leaves. In addition, overexpression of NbBiP4 or NbCRT3 enhanced the targeting of BaMV TGBp1 to plasmodesmata (PD), indicating that NbBiP4 and NbCRT3 interact with TGBp3 to promote the intracellular transport of virion cargo to PD that facilitates virus cell-to-cell movement. Our findings revealed additional roles for NbBiP4 and NbCRT3 in BaMV intracellular movement through ER networks or ER-derived vesicles to PD, which enhances the spread of BaMV in N. benthamiana.

Development of a tag-free plant-made interferon gamma production system with improved therapeutic efficacy against viruses.

Plants offer a promising platform for cost-effective production of biologically active therapeutic glycoproteins. In previous studies, we have developed a plant expression system based on Bamboo mosaic virus (BaMV) by incorporating secretory signals and an affinity tag, which resulted in notably enhanced yields of soluble and secreted fusion glycoproteins (FGs) in Nicotiana benthamiana. However, the presence of fusion tags on recombinant glycoproteins is undesirable for biomedical applications. This study aimed to develop a refined expression system that can efficiently produce tag-free glycoproteins in plants, with enhanced efficacy of mature interferon gamma (mIFNγ) against viruses. To accommodate the specific requirement of different target proteins, three enzymatically or chemically cleavable linkers were provided in this renovated BaMV-based expression system. We demonstrated that Tobacco etch virus (TEV) protease could process the specific cleavage site (LTEV) of the fusion protein, designated as SSExtHis(SP)10LTEV-mIFNγ, with optimal efficiency under biocompatible conditions to generate tag-free mIFNγ glycoproteins. The TEV protease and secretory-affinity tag could be effectively removed from the target mIFNγ glycoproteins through Ni2+-NTA chromatography. In addition, the result of an antiviral assay showed that the tag-free mIFNγ glycoproteins exhibited enhanced biological properties against Sindbis virus, with comparable antiviral activity of the commercialized HEK293-expressed hIFNγ. Thus, the improved BaMV-based expression system developed in this study may provide an alternative strategy for producing tag-free therapeutic glycoproteins intended for biomedical applications.

Characterization of Virus-Inducible Orchid Argonaute 5b Promoter and Its Functional Characterization in Nicotiana benthamiana during Virus Infection.

Plant ARGONAUTES (AGOs) play a significant role in the defense against viral infection. Previously, we have demonstrated that AGO5s encoded in Phalaenopsis aphrodite subsp. formosana (PaAGO5s) took an indispensable part in defense against major viruses. To understand the underlying defense mechanism, we cloned PaAGO5s promoters (pPaAGO5s) and analyzed their activity in transgenic Nicotiana benthamiana using ß-glucuronidase (GUS) as a reporter gene. GUS activity analyses revealed that during Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) infections, pPaAGO5b activity was significantly increased compared to pPaAGO5a and pPaAGO5c. Analysis of pPaAGO5b 5'-deletion revealed that pPaAGO5b_941 has higher activity during virus infection. Further, yeast one-hybrid analysis showed that the transcription factor NbMYB30 physically interacted with pPaAGO5b_941 to enhance its activity. Overexpression and silencing of NbMYB30 resulted in up- and downregulation of GUS expression, respectively. Exogenous application and endogenous measurement of phytohormones have shown that methyl jasmonate and salicylic acid respond to viral infections. NbMYB30 overexpression and its closest related protein, PaMYB30, in P. aphrodite subsp. formosana reduced CymMV accumulation in P. aphrodite subsp. formosana. Based on these discoveries, this study uncovers the interaction between virus-responsive promoter and the corresponding transcription factor in plants.

NbNAC42 and NbZFP3 Transcription Factors Regulate the Virus Inducible NbAGO5 Promoter in Nicotiana benthamiana.

Plant argonautes (AGOs) play important roles in the defense responses against viruses. The expression of Nicotiana benthamiana AGO5 gene (NbAGO5) is highly induced by Bamboo mosaic virus (BaMV) infection; however, the underlying mechanisms remain elusive. In this study, we have analyzed the potential promoter activities of NbAGO5 and its interactions with viral proteins by using a 2,000 bp fragment, designated as PN1, upstream to the translation initiation of NbAGO5. PN1 and seven serial 5'-deletion mutants (PN2-PN8) were fused with a ß-glucuronidase (GUS) reporter and introduced into the N. benthamiana genome by Agrobacterium-mediated transformation for further characterization. It was found that PN4-GUS transgenic plants were able to drive strong GUS expression in the whole plant. In the virus infection tests, the GUS activity was strongly induced in PN4-GUS transgenic plants after being challenged with potexviruses. Infiltration of the transgenic plants individually with BaMV coat protein (CP) or triple gene block protein 1 (TGBp1) revealed that only TGBp1 was crucial for inducing the NbAGO5 promoter. To identify the factors responsible for controlling the activity of the NbAGO5 promoter, we employed yeast one-hybrid screening on a transcription factor cDNA library. The result showed that NbNAC42 and NbZFP3 could directly bind the 704 bp promoter regions of NbAGO5. By using overexpressing and virus-induced gene silencing techniques, we found that NbNAC42 and NbZFP3 regulated and downregulated, respectively, the expression of the NbAGO5 gene. Upon virus infection, NbNAC42 played an important role in regulating the expression of NbAGO5. Together, these results provide new insights into the modulation of the defense mechanism of N. benthamiana against viruses. This virus inducible promoter could be an ideal candidate to drive the target gene expression that could improve the anti-virus abilities of crops in the future.

The gibberellic acid derived from the plastidial MEP pathway is involved in the accumulation of Bamboo mosaic virus.

A gene upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) infection was revealed as 1-deoxy-d-xylulose-5-phosphate reductoisomerase (NbDXR). DXR is the key enzyme in the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway that catalyzes the conversion of 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol-4-phosphate. Knockdown and overexpression of NbDXR followed by BaMV inoculation revealed that NbDXR is involved in BaMV accumulation. Treating leaves with fosmidomycin, an inhibitor of DXR function, reduced BaMV accumulation. Subcellular localization confirmed that DXR is a chloroplast-localized protein by confocal microscopy. Furthermore, knockdown of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, one of the enzymes in the MEP pathway, also reduced BaMV accumulation. The accumulation of BaMV increased significantly in protoplasts treated with isopentenyl pyrophosphate. Thus, the metabolites of the MEP pathway could be involved in BaMV infection. To identify the critical components involved in BaMV accumulation, we knocked down the crucial enzyme of isoprenoid synthesis, NbGGPPS11 or NbGGPPS2. Only NbGGPPS2 was involved in BaMV infection. The geranylgeranyl pyrophosphate (GGPP) synthesized by NbGGPPS2 is known for gibberellin synthesis. We confirmed this result by supplying gibberellic acid exogenously on leaves, which increased BaMV accumulation. The de novo synthesis of gibberellic acid could assist BaMV accumulation.

Special Issue "State-of-the-Art Plant-Virus Interactions in Asia".

As rivals over the long history of co-evolution, viruses and host plants have each developed specialized strategies and machineries to cope with the rivalry [...].

First Report of Distinct Bamboo mosaic virus (BaMV) Isolates Infecting Bambusa funghomii in Vietnam and the Identification of a Highly Variable Region in the BaMV Genome.

New isolates of the Bamboo mosaic virus (BaMV) were identified in Bambusa funghomii bamboo in Vietnam. Sequence analyses revealed that the Vietnam isolates are distinct from all known BaMV strains, sharing the highest sequence identities (about 77%) with the Yoshi isolates reported in California, USA. Unique satellite RNAs were also found to be associated with the BaMV Vietnam isolates. A possible recombination event was detected in the genome of BaMV-VN2. A highly variable region was identified in the ORF1 gene, in between the methyl transferase domain and helicase domain. These results revealed the presence of unique BaMV isolates in an additional bamboo species in one more country, Vietnam, and provided evidence in support of the possible involvement of environmental or host factors in the diversification and evolution of BaMV.

Cloning and Organelle Expression of Bamboo Mitochondrial Complex I Subunits Nad1, Nad2, Nad4, and Nad5 in the Yeast Saccharomyces cerevisiae.

Mitochondrial respiratory complex I catalyzes electron transfer from NADH to ubiquinone and pumps protons from the matrix into the intermembrane space. In particular, the complex I subunits Nad1, Nad2, Nad4, and Nad5, which are encoded by the nad1, nad2, nad4, and nad5 genes, reside at the mitochondrial inner membrane and possibly function as proton (H+) and ion translocators. To understand the individual functional roles of the Nad1, Nad2, Nad4, and Nad5 subunits in bamboo, each cDNA of these four genes was cloned into the pYES2 vector and expressed in the mitochondria of the yeast Saccharomyces cerevisiae. The mitochondrial targeting peptide mt gene (encoding MT) and the egfp marker gene (encoding enhanced green fluorescent protein, EGFP) were fused at the 5'-terminal and 3'-terminal ends, respectively. The constructed plasmids were then transformed into yeast. RNA transcripts and fusion protein expression were observed in the yeast transformants. Mitochondrial localizations of the MT-Nad1-EGFP, MT-Nad2-EGFP, MT-Nad4-EGFP, and MT-Nad5-EGFP fusion proteins were confirmed by fluorescence microscopy. The ectopically expressed bamboo subunits Nad1, Nad2, Nad4, and Nad5 may function in ion translocation, which was confirmed by growth phenotype assays with the addition of different concentrations of K+, Na+, or H+.

MiR398-regulated antioxidants contribute to Bamboo mosaic virus accumulation and symptom manifestation.

Virus infections that cause mosaic or mottling in leaves commonly also induce increased levels of reactive oxygen species (ROS). However, how ROS contributes to symptoms is less well documented. Bamboo mosaic virus (BaMV) causes chlorotic mosaic symptoms in both Brachypodium distachyon and Nicotiana benthamiana. The BaMV △CPN35 mutant with an N-terminal deletion of its coat protein gene exhibits asymptomatic infection independently of virus titer. Histochemical staining of ROS in mock-, BaMV-, and BaMV△CPN35-infected leaves revealed that hydrogen peroxide (H2O2) accumulated solely in BaMV-induced chlorotic spots. Moreover, exogenous H2O2 treatment enhanced yellowish chlorosis in BaMV-infected leaves. Both BaMV and BaMV△CPN35 infection could induce the expression of Cu/Zu superoxide dismutase (CSD) antioxidants at messenger RNA and protein level. However, BaMV triggered the abundant accumulation of full-length NbCSD2 preprotein (prNbCSD2, without transit peptide cleavage), whereas BaMV△CPN35 induced a truncated prNbCSD2. Confocal microscopy showed that majority of NbCSD2-green fluorescent protein (GFP) predominantly localized in the cytosol upon BaMV infection, but BaMV△CPN35 infection tended to cause NbCSD2-GFP to remain in chloroplasts. By 5'-RNA ligase-mediated rapid amplification of cDNA ends, we validated CSDs are the targets of miR398 in vivo. Furthermore, BaMV infection increased the level of miR398, while the level of BaMV titer was regulated positively by miR398 but negatively by CSD2. In contrast, overexpression of cytosolic form NbCSD2, impairing the transport into chloroplasts, greatly enhanced BaMV accumulation. Taken together, our results indicate that induction of miR398 by BaMV infection may facilitate viral titer accumulation, and cytosolic prNbCSD2 induction may contribute to H2O2 accumulation, resulting in the development of BaMV chlorotic symptoms in plants.

Voltage-dependent anion channel proteins associate with dynamic Bamboo mosaic virus-induced complexes.

Infection cycles of viruses are highly dependent on membrane-associated host factors. To uncover the infection cycle of Bamboo mosaic virus (BaMV) in detail, we purified the membrane-associated viral complexes from infected Nicotiana benthamiana plants and analyzed the involved host factors. Four isoforms of voltage-dependent anion channel (VDAC) proteins on the outer membrane of mitochondria were identified due to their upregulated expression in the BaMV complex-enriched membranous fraction. Results from loss- and gain-of-function experiments indicated that NbVDAC2, -3, and -4 are essential for efficient BaMV accumulation. During BaMV infection, all NbVDACs concentrated into larger aggregates, which overlapped and trafficked with BaMV virions to the structure designated as the "dynamic BaMV-induced complex." Besides the endoplasmic reticulum and mitochondria, BaMV replicase and double-stranded RNAs were also found in this complex, suggesting the dynamic BaMV-induced complex is a replication complex. Yeast two-hybrid and pull-down assays confirmed that BaMV triple gene block protein 1 (TGBp1) could interact with NbVDACs. Confocal microscopy revealed that TGBp1 is sufficient to induce NbVDAC aggregates, which suggests that TGBp1 may play a pivotal role in the NbVDAC-virion complex. Collectively, these findings indicate that NbVDACs may associate with the dynamic BaMV-induced complex via TGBp1 and NbVDAC2, -3, or -4 and can promote BaMV accumulation. This study reveals the involvement of mitochondrial proteins in a viral complex and virus infection.

The function of chloroplast ferredoxin-NADP+ oxidoreductase positively regulates the accumulation of bamboo mosaic virus in Nicotiana benthamiana.

A gene down-regulated in Nicotiana benthamiana after bamboo mosaic virus (BaMV) infection had high identity to the nuclear-encoded chloroplast ferredoxin NADP+ oxidoreductase gene (NbFNR). NbFNR is a flavoenzyme involved in the photosynthesis electron transport chain, catalysing the conversion of NADP+ into NADPH. To investigate whether NbFNR is involved in BaMV infection, we used virus-induced gene silencing to reduce the expression of NbFNR in leaves and protoplasts. After BaMV inoculation, the accumulation of BaMV coat protein and RNA was significantly reduced. The transient expression of NbFNR fused with orange fluorescent protein (OFP) localized in the chloroplasts and elevated the level of BaMV coat protein. These results suggest that NbFNR could play a positive role in regulating BaMV accumulation. Expressing a mutant that failed to translocate to the chloroplast did not assist in BaMV accumulation. Another mutant with a catalytic site mutation could support BaMV accumulation to some extent, but accumulation was significantly lower than that of the wild type. In an in vitro replication assay, the replicase complex with FNR inhibitor, heparin, the RdRp activity was reduced. Furthermore, BaMV replicase was revealed to interact with NbFNR in yeast two-hybrid and co-immunoprecipitation experiments. Overall, these results suggest that NbFNR localized in the chloroplast with functional activity could efficiently assist BaMV accumulation.

Convenient Auto-Processing Vector Based on Bamboo Mosaic Virus for Presentation of Antigens Through Enzymatic Coupling.

We have developed a new binary epitope-presenting CVP platform based on bamboo mosaic virus (BaMV) by using the sortase A (SrtA)-mediated ligation technology. The reconstructed BaMV genome harbors two modifications: 1) a coat protein (CP) with N-terminal extension of the tobacco etch virus (TEV) protease recognition site plus 4 extra glycine (G) residues as the SrtA acceptor; and 2) a TEV protease coding region replacing that of the triple-gene-block proteins. Inoculation of such construct, pKB5G, on Nicotiana benthamiana resulted in the efficient production of filamentous CVPs ready for SrtA-mediated ligation with desired proteins. The second part of the binary platform includes an expression vector for the bacterial production of donor proteins. We demonstrated the applicability of the platform by using the recombinant envelope protein domain III (rEDIII) of Japanese encephalitis virus (JEV) as the antigen. Up to 40% of the BaMV CP subunits in each CVP were loaded with rEDIII proteins in 1 min. The rEDIII-presenting BaMV CVPs (BJLPET5G) could be purified using affinity chromatography. Immunization assays confirmed that BJLPET5G could induce the production of neutralizing antibodies against JEV infections. The binary platform could be adapted as a useful alternative for the development and mass production of vaccine candidates.

NbPsbO1 Interacts Specifically with the Bamboo Mosaic Virus (BaMV) Subgenomic RNA (sgRNA) Promoter and Is Required for Efficient BaMV sgRNA Transcription.

Many positive-strand (+) RNA viruses produce subgenomic RNAs (sgRNAs) in the infection cycle through the combined activities of viral replicase and host proteins. However, knowledge about host proteins involved in direct sgRNA promoter recognition is limited. Here, in the partially purified replicase complexes from Bamboo mosaic virus (BaMV)-infected tissue, we have identified the Nicotiana benthamiana photosystem II oxygen-evolving complex protein, NbPsbO1, which specifically interacted with the promoter of sgRNA but not that of genomic RNA (gRNA). Silencing of NbPsbO1 expression suppressed BaMV accumulation in N. benthamiana protoplasts without affecting viral gRNA replication. Overexpression of wild-type NbPsbO1 stimulated BaMV sgRNA accumulation. Fluorescent microscopy examination revealed that the fluorescence associated with NbPsbO1 was redistributed from chloroplast granal thylakoids to stroma in BaMV-infected cells. Overexpression of a mislocalized mutant of NbPsbO1, dTPPsbO1-T7, inhibited BaMV RNA accumulation in N. benthamiana, whereas overexpression of an NbPsbO1 derivative, sPsbO1-T7, designed to be targeted to chloroplast stroma, upregulated the sgRNA level. Furthermore, depletion of NbPsbO1 in BaMV RdRp preparation significantly inhibited sgRNA synthesis in vitro but exerted no effect on (+) or (-) gRNA synthesis, which indicates that NbPsbO1 is required for efficient sgRNA synthesis. These results reveal a novel role for NbPsbO1 in the selective enhancement of BaMV sgRNA transcription, most likely via direct interaction with the sgRNA promoter. IMPORTANCE Production of subgenomic RNAs (sgRNAs) for efficient translation of downstream viral proteins is one of the major strategies adapted for viruses that contain a multicistronic RNA genome. Both viral genomic RNA (gRNA) replication and sgRNA transcription rely on the combined activities of viral replicase and host proteins, which recognize promoter regions for the initiation of RNA synthesis. However, compared to the cis-acting elements involved in the regulation of sgRNA synthesis, the host factors involved in sgRNA promoter recognition mostly remain to be elucidated. Here, we found a chloroplast protein, NbPsbO1, which specifically interacts with Bamboo mosaic virus (BaMV) sgRNA promoter. We showed that NbPsbO1 is relocated to the BaMV replication site in BaMV-infected cells and demonstrated that NbPsbO1 is required for efficient BaMV sgRNA transcription but exerts no effect on gRNA replication. This study provides a new insight into the regulating mechanism of viral gRNA and sgRNA synthesis.

Exploring the Multifunctional Roles of Odontoglossum Ringspot Virus P126 in Facilitating Cymbidium Mosaic Virus Cell-to-Cell Movement during Mixed Infection.

Synergistic interactions among viruses, hosts and/or transmission vectors during mixed infection can alter viral titers, symptom severity or host range. Viral suppressors of RNA silencing (VSRs) are considered one of such factors contributing to synergistic responses. Odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV), which are two of the most significant orchid viruses, exhibit synergistic symptom intensification in Phalaenopsis orchids with unilaterally enhanced CymMV movement by ORSV. In order to reveal the underlying mechanisms, we generated infectious cDNA clones of ORSV and CymMV isolated from Phalaenopsis that exerted similar unilateral synergism in both Phalaenopsis orchid and Nicotiana benthamiana. Moreover, we show that the ORSV replicase P126 is a VSR. Mutagenesis analysis revealed that mutation of the methionine in the carboxyl terminus of ORSV P126 abolished ORSV replication even though some P126 mutants preserved VSR activity, indicating that the VSR function of P126 alone is not sufficient for viral replication. Thus, P126 functions in both ORSV replication and as a VSR. Furthermore, P126 expression enhanced cell-to-cell movement and viral titers of CymMV in infected Phalaenopsis flowers and N. benthamiana leaves. Taking together, both the VSR and protein function of P126 might be prerequisites for unilaterally enhancing CymMV cell-to-cell movement by ORSV.