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Melampsora larici-populina (Mlp) is a devastating pathogen of poplar trees, causing the defoliating poplar leaf rust disease. Genomic studies have revealed that Mlp possesses a repertoire of 1184 small secreted proteins (SSPs), some of them being characterized as candidate effectors. However, how they promote virulence is still unclear. This study investigates the candidate effector Mlp37347's role during infection. We developed a stable Arabidopsis transgenic line expressing Mlp37347 tagged with the green fluorescent protein (GFP). We found that the effector accumulated exclusively at plasmodesmata (PD). Moreover, the presence of the effector at plasmodesmata favors enhanced plasmodesmatal flux and reduced callose deposition. Transcriptome profiling and a gene ontology (GO) analysis of transgenic Arabidopsis plants expressing the effector revealed that the genes involved in glucan catabolic processes are up-regulated. This effector has previously been shown to interact with glutamate decarboxylase 1 (GAD1), and in silico docking analysis supported the strong binding between Mlp37347 and GAD1 in this study. In infection assays, the effector promoted Hyalonoperospora arabidopsidis growth but not bacterial growth. Our investigation suggests that the effector Mlp37347 targets PD in host cells and promotes parasitic growth.

Turnip mosaic virus (TuMV) reorganizes the endomembrane system of the infected cell to generate endoplasmic-reticulum-derived motile vesicles containing viral replication complexes. The membrane-associated viral protein 6K 2 plays a key role in the formation of these vesicles. Using confocal microscopy, we observed that this viral protein, a marker for viral replication complexes, localized in the extracellular space of infected Nicotiana benthamiana leaves. Previously, we showed that viral RNA is associated with multivesicular bodies (MVBs). Here, using transmission electron microscopy, we observed the proliferation of MVBs during infection and their fusion with the plasma membrane that resulted in the release of their intraluminal vesicles in the extracellular space. Immunogold labeling with a monoclonal antibody that recognizes double-stranded RNA indicated that the released vesicles contained viral RNA. Focused ion beam-extreme high-resolution scanning electron microscopy was used to generate a three-dimensional image that showed extracellular vesicles in the cell wall. The presence of TuMV proteins in the extracellular space was confirmed by proteomic analysis of purified extracellular vesicles from N benthamiana and Arabidopsis ( Arabidopsis thaliana ). Host proteins involved in biotic defense and in interorganelle vesicular exchange were also detected. The association of extracellular vesicles with viral proteins and RNA emphasizes the implication of the plant extracellular space in viral infection., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Like other positive-strand RNA viruses, the Turnip mosaic virus (TuMV) infection leads to the formation of viral vesicles at the endoplasmic reticulum (ER). Once released from the ER, the viral vesicles mature intracellularly and then move intercellularly. While it is known that the membrane-associated viral protein 6K2 plays a role in the process, the contribution of host proteins has been poorly defined. In this article, we show that 6K2 interacts with RHD3, an ER fusogen required for efficient ER fusion. When RHD3 is mutated, a delay in the development of TuMV infection is observed. We found that the replication of TuMV and the cell-to-cell movement of its replication vesicles are impaired in rhd3 This defect can be tracked to a delayed maturation of the viral vesicles from the replication incompetent to the competent state. Furthermore, 6K2 can relocate RHD3 from the ER to viral vesicles. However, a Golgi-localized mutated 6K2 GV is unable to interact and relocate RHD3 to viral vesicles. We conclude that the maturation of TuMV replication vesicles requires RHD3 for efficient viral replication and movement., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Infection of plant cells by RNA viruses leads to the generation of organelle-like subcellular structures that contain the viral replication complex. During Turnip mosaic virus (TuMV) infection of Nicotiana benthamiana , the viral membrane protein 6K 2 plays a key role in the release of motile replication vesicles from the host endoplasmic reticulum (ER). Here, we demonstrate that 6K 2 contains a GxxxG motif within its predicted transmembrane domain that is vital for TuMV infection. Replacement of the Gly with Val within this motif inhibited virus production, and this was due to a relocation of the viral protein to the Golgi apparatus and the plasma membrane. This indicated that passage of 6K 2 through the Golgi apparatus is a dead-end avenue for virus infection. Impairing the fusion of transport vesicles between the ER and the Golgi apparatus by overexpression of the SNARE Sec22 protein resulted in enhanced intercellular virus movement. Likewise, expression of nonfunctional, Golgi-located synaptotagmin during infection enhanced TuMV intercellular movement. 6K 2 copurified with VTI11, a prevacuolar compartment SNARE protein. An Arabidopsis thaliana vti11 mutant was completely resistant to TuMV infection. We conclude that TuMV replication vesicles bypass the Golgi apparatus and take an unconventional pathway that may involve prevacuolar compartments/multivesicular bodies for virus infection., (© 2018 American Society of Plant Biologists. All rights reserved.)

Positive-sense (+) RNA viruses represent the most abundant group of viruses and are dependent on the host cell machinery to replicate. One remarkable feature that occurs after (+) RNA virus entry into cells is the remodeling of host endomembranes, leading to the formation of viral replication factories. Recently, rapid progress in three-dimensional (3D) imaging technologies, such as electron tomography (ET) and focused ion beam-scanning electron microscopy (FIB-SEM), has enabled researchers to visualize the novel membrane structures induced by viruses at high resolution. These 3D imaging technologies provide new mechanistic insights into the viral infection cycle. In this review, we summarize the latest reports on the cellular remodeling that occurs during plant virus infection; in particular, we focus on studies that provide 3D architectural information on viral replication factories. We also outline the mechanisms underlying the formation of these membranous structures and discuss possible future research directions.

Plant viruses move from the initially infected cell to adjacent cells through plasmodesmata (PDs). To do so, viruses encode dedicated protein(s) that facilitate this process. How viral proteins act together to support the intercellular movement of viruses is poorly defined. Here, by using an infection-free intercellular vesicle movement assay, we investigate the action of CI (cylindrical inclusion) and P3N-PIPO (amino-terminal half of P3 fused to Pretty Interesting Potyviridae open reading frame), the two PD-localized potyviral proteins encoded by Turnip mosaic virus (TuMV), in the intercellular movement of the viral replication vesicles. We provide evidence that CI and P3N-PIPO are sufficient to support the PD targeting and intercellular movement of TuMV replication vesicles induced by 6K2, a viral protein responsible for the generation of replication vesicles. 6K2 interacts with CI but not P3N-PIPO. When this interaction is impaired, the intercellular movement of TuMV replication vesicles is inhibited. Furthermore, in transmission electron microscopy, vesicular structures are observed in connection with the cylindrical inclusion bodies at structurally modified PDs in cells coexpressing 6K2, CI, and P3N-PIPO. CI is directed to PDs through its interaction with P3N-PIPO. We hypothesize that CI serves as a docking point for PD targeting and the intercellular movement of TuMV replication vesicles. This work contributes to a better understanding of the roles of different viral proteins in coordinating the intercellular movement of viral replication vesicles., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Porcine parvoviruses (PPV) are known to be particularly resistant to many disinfectants used to control other non-enveloped viruses. However, effective disinfectants used against PPV are harsh and corrosive to animal health facilities and the environment. We propose a noncorrosive "green" disinfectant that generates peracetic acid in-situ and is capable of inactivating PPV completely at a 1% concentration for a 10-minute contact time.

Unlabelled: Positive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K2 plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2 tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation., Importance: Positive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The ultrastructure of turnip mosaic virus (TuMV)-induced membrane remodeling was investigated over several days of infection. The first change that was observed involved endoplasmic reticulum-connected convoluted membrane accumulation. This was followed by the formation of single-membrane tubules, which were shown to be viral RNA replication sites. Later in the infection process, double-membrane tubular structures were observed and were associated with viral particle bundles. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. This work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Positive-sense RNA viruses remodel host cell endomembranes to generate quasi-organelles known as "viral factories" to coordinate diverse viral processes, such as genome translation and replication. It is also becoming clear that enclosing viral RNA (vRNA) complexes within membranous structures is important for virus cell-to-cell spread throughout the host. In plant cells infected by turnip mosaic virus (TuMV), a member of the family Potyviridae, peripheral motile endoplasmic reticulum (ER)-derived viral vesicles are produced that carry the vRNA to plasmodesmata for delivery into adjacent noninfected cells. The viral protein 6K2 is responsible for the formation of these vesicles, but how 6K2 is involved in their biogenesis is unknown. We show here that 6K2 is associated with cellular membranes. Deletion mapping and site-directed mutagenesis experiments defined a soluble N-terminal 12-amino-acid stretch, in particular a potyviral highly conserved tryptophan residue and two lysine residues that were important for vesicle formation. When the tryptophan residue was changed into an alanine in the viral polyprotein, virus replication still took place, albeit at a reduced level, but cell-to-cell movement of the virus was abolished. Yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitation experiments showed that 6K2 interacted with Sec24a, a COPII coatomer component. Appropriately, TuMV systemic movement was delayed in an Arabidopsis thaliana mutant line defective in Sec24a. Intercellular movement of TuMV replication vesicles thus requires ER export of 6K2, which is mediated by the interaction of the N-terminal domain of the viral protein with Sec24a., Importance: Many plant viruses remodel the endoplasmic reticulum (ER) to generate vesicles that are associated with the virus replication complex. The viral protein 6K2 of turnip mosaic virus (TuMV) is known to induce ER-derived vesicles that contain vRNA as well as viral and host proteins required for vRNA synthesis. These vesicles not only sustain vRNA synthesis, they are also involved in the intercellular trafficking of vRNA. In this investigation, we found that the N-terminal soluble domain of 6K2 is required for ER export of the protein and for the formation of vesicles. ER export is not absolutely required for vRNA replication but is necessary for virus cell-to-cell movement. Furthermore, we found that 6K2 physically interacts with the COPII coatomer Sec24a and that an Arabidopsis thaliana mutant line with a defective Sec24a shows a delay in the systemic infection by TuMV., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Plant viruses move systemically in plants through the phloem. They move as virions or as ribonucleic protein complexes, although it is not clear what these complexes are made of. The approximately 10-kb RNA genome of Turnip mosaic virus (TuMV) encodes a membrane protein, known as 6K2, that induces endomembrane rearrangements for the formation of viral replication factories. These factories take the form of vesicles that contain viral RNA (vRNA) and viral replication proteins. In this study, we report the presence of 6K2-tagged vesicles containing vRNA and the vRNA-dependent RNA polymerase in phloem sieve elements and in xylem vessels. Transmission electron microscopy observations showed the presence in the xylem vessels of vRNA-containing vesicles that were associated with viral particles. Stem-girdling experiments, which leave xylem vessels intact but destroy the surrounding tissues, confirmed that TuMV could establish a systemic infection of the plant by going through xylem vessels. Phloem sieve elements and xylem vessels from Potato virus X-infected plants also contained lipid-associated nonencapsidated vRNA, indicating that the presence of membrane-associated ribonucleic protein complexes in the phloem and xylem may not be limited to TuMV. Collectively, these studies indicate that viral replication factories could end up in the phloem and the xylem., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

It is generally accepted that in order to establish a systemic infection in a plant, viruses move from the initially infected cell to the vascular tissues by cell-to-cell movement through plasmodesmata (PD), and load into the vascular conducting tubes (i.e. phloem sieve elements and xylem vessel elements) for long-distance movement. The viral unit in these movements can be a virion or a yet-to-be-defined ribonucleic protein (RNP) complex. Using live-cell imaging, our laboratory has previously demonstrated that membrane-bound replication complexes move cell-to-cell during turnip mosaic virus (TuMV) infection. Our recent study shows that these membrane-bound replication complexes end up in the vascular conducting tubes, which is likely the case for potato virus X (PVX) also. The presence of TuMV-induced membrane complexes in xylem vessels suggests that viral components could also be found in other apoplastic regions of the plant, such as the intercellular space. This possibility may have implications regarding how we approach the study of plant innate immune responses against viruses.

Plant viruses, like animal viruses, induce the formation of novel intracellular membranous structures that provide an optimum environment for coordinating diverse viral processes such as viral RNA synthesis and virus egress. Membrane reshaping is accomplished by the expression of specific membrane-associated viral proteins that interact with host proteins involved in membrane trafficking processes. Plant virus-induced membranous structures are motile, and this intracellular motility is required for the transport of viral RNA from sites of synthesis to plasmodesmata, which are used to move viral RNA from cell to cell. Cellular movement of these virus-induced bodies requires myosin motor activity and is dependent on the secretory pathway. The coupling of membrane-associated replication complexes with virus intra- and intercellular trafficking may explain why viral infection of neighboring cells is established rapidly and efficiently.

To successfully infect plants, viruses replicate in an initially infected cell and then move to neighboring cells through plasmodesmata (PDs). However, the nature of the viral entity that crosses over the cell barrier into non-infected ones is not clear. The membrane-associated 6K2 protein of turnip mosaic virus (TuMV) induces the formation of vesicles involved in the replication and intracellular movement of viral RNA. This study shows that 6K2-induced vesicles trafficked toward the plasma membrane and were associated with plasmodesmata (PD). We demonstrated also that 6K2 moved cell-to-cell into adjoining cells when plants were infected with TuMV. 6K2 was then fused to photo-activable GFP (6K2:PAGFP) to visualize how 6K2 moved intercellularly during TuMV infection. After activation, 6K2:PAGFP-tagged vesicles moved to the cell periphery and across the cell wall into adjacent cells. These vesicles were shown to contain the viral RNA-dependent RNA polymerase and viral RNA. Symplasmic movement of TuMV may thus be achieved in the form of a membrane-associated viral RNA complex induced by 6K2.

The plant cell is reprogrammed and undergoes drastic morphological alterations during infection by viruses. Infection leads to the formation of viral factories, derived from host cell membranes for viral replication. This review discusses the biogenesis of the different viral replication factories that are observed and the impact of their formation on the cell metabolism. The involvement of viral factories in cell-to-cell movement of the virus and modifications of plasmodesmata are also described.

The secretory pathway of eukaryotic cells has an elaborated set of endomembrane compartments involved in the synthesis, modification, and sorting of proteins and lipids. The secretory pathway in plant cells shares many features with that in other eukaryotic cells but also has distinct characteristics important for fundamental cell and developmental processes and for proper immune responses. Recently, there has been evidence that the remodeling of this pathway, and often the formation of viral-induced organelles, play an important role in viral replication and spread. The modification of the host secretory pathway seems to be a common feature among most single-stranded positive ss(+)RNA and even some DNA viruses. In this review, we will present the recent advances in the understanding of the organization and dynamics of the plant secretory pathway and the molecular regulation of membrane trafficking in the pathway. We will also discuss how different plant viruses may interact with the host secretory pathway for their efficient replication and spread, with a focus on tobacco mosaic virus and turnip mosaic virus.

The contribution of different host cell transport systems in the intercellular movement of turnip mosaic virus (TuMV) was investigated. To discriminate between primary infections and secondary infections associated with the virus intercellular movement, a gene cassette expressing GFP-HDEL was inserted adjacent to a TuMV infectious cassette expressing 6K₂:mCherry, both within the T-DNA borders of the binary vector pCambia. In this system, both gene cassettes were delivered to the same cell by a single binary vector and primary infection foci emitted green and red fluorescence while secondarily infected cells emitted only red fluorescence. Intercellular movement was measured at 72 hours post infiltration and was estimated to proceed at an average rate of one cell being infected every three hours over an observation period of 17 hours. To determine if the secretory pathway were important for TuMV intercellular movement, chemical and protein inhibitors that blocked both early and late secretory pathways were used. Treatment with Brefeldin A or Concanamycin A or expression of ARF1 or RAB-E1d dominant negative mutants, all of which inhibit pre- or post-Golgi transport, reduced intercellular movement by the virus. These treatments, however, did not inhibit virus replication in primary infected cells. Pharmacological interference assays using Tyrphostin A23 or Wortmannin showed that endocytosis was not important for TuMV intercellular movement. Lack of co-localization by endocytosed FM4-64 and Ara7 (AtRabF2b) with TuMV-induced 6K₂-tagged vesicles further supported this conclusion. Microfilament depolymerizing drugs and silencing expression of myosin XI-2 gene, but not myosin VIII genes, also inhibited TuMV intercellular movement. Expression of dominant negative myosin mutants confirmed the role played by myosin XI-2 as well as by myosin XI-K in TuMV intercellular movement. Using this dual gene cassette expression system and transport inhibitors, components of the secretory and actomyosin machinery were shown to be important for TuMV intercellular spread.

Although there is a significant amount of literature that deals with the identification of plant viral proteins involved in membrane remodeling and vesicle production in infected cells, there are very few investigations that report on the impact that infection has on the overall architecture and dynamics of the early secretory endomembranes. Recent investigations have shown that for some viruses the endoplasmic reticulum, Golgi bodies and other organelles are heavily recruited into virus-induced perinuclear structures. These structures are not isolated organelles and are dynamically connected to the bulk of non-modified endomembranes. They also have a functional link with peripheral motile vesicles involved in virus intracellular movement. The full molecular events that consubstantiate with this endomembrane recruitment in virus-induced structures remain to be elucidated but viral genome replication and virion assembly are probably taking place within these structures., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The impact of turnip mosaic virus (TuMV) infection on the endomembranes of the host early secretory pathway was investigated using an infectious clone that has been engineered for tagging viral membrane structures with a fluorescent protein fused to the viral protein 6K(2). TuMV infection led to the amalgamation of the endoplasmic reticulum (ER), Golgi apparatus, COPII coatamers, and chloroplasts into a perinuclear globular structure that also contained viral proteins. One consequence of TuMV infection was that protein secretion was blocked at the ER-Golgi interface. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the perinuclear structure cannot be restocked in viral components but was dynamically connected to the bulk of the Golgi apparatus and the ER. Experiments with 6K(2) fused to photoactivable green fluorescent protein (GFP) showed that production of motile peripheral 6K(2) vesicles was functionally linked to the perinuclear structure. Disruption of the early secretory pathway did not prevent the formation of the perinuclear globular structure, enhanced the clustering of peripheral 6K(2) vesicles with COPII coatamers, and led to inhibition of cell-to-cell virus movement. This suggests that a functional secretory pathway is not required for the formation of the TuMV perinuclear globular structure and peripheral vesicles but is needed for successful viral intercellular propagation.

For some plant positive-sense RNA viruses, a protein known as VPg (short for virus protein, genome linked) is covalently linked to the 5' end of the viral RNA. The VPg is an intrinsically disordered protein, and this property would confer an ability to bind several proteins. Accordingly, the potyvirus VPg interacts with many proteins, notably host factors involved in protein synthesis within viral replication factories or within the nucleus. The number of protein partners, the clustering of the various interactions centering around it, the biological importance for some of these interactions (e.g. VPg-eIF4E) and the intrinsically disordered state of the protein are all elements that support the notion that VPg is a hub protein that controls many processes leading to virus production and spread., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nicotiana benthamiana plants were agroinfiltrated with an infectious clone of the Turnip mosaic virus (TuMV) that was engineered to tag replication vesicles with either GFP or mCherry fluorescent proteins. Punctuate vesicle structures were observed in the cytoplasm of infected cells corresponding to viral replication factories. The vesicles were highly motile and co-aligned with the microfilaments. Utilization of latrunculin B, an inhibitor of microfilament polymerization, reduced accumulation of the virus, suggesting that microfilaments are necessary during infection. To investigate biogenesis of the vesicles, leaves were infected simultaneously with two recombinant TuMV infectious clones, one that labeled vesicles in red and one that labeled them in green. We observed cell with green only and red only vesicles indicating a single viral genome origin. In some cases, vesicles exhibited sectors of green, red and yellow fluorescence were also observed, demonstrating that fusion among individual vesicles is possible. Based on those results we propose a model for the biogenesis of viral factory, where viral translation and replication are tightly coupled within virus-induced vesicles.

This review focuses on the extensive membrane and organelle rearrangements that have been observed in plant cells infected with RNA viruses. The modifications generally involve the formation of spherules, vesicles, and/or multivesicular bodies associated with various organelles such as the endoplasmic reticulum and peroxisomes. These virus-induced organelles house the viral RNA replication complex and are known as virus factories or viroplasms. Membrane and organelle alterations are attributed to the action of one or two viral proteins, which additionally act as a scaffold for the assembly of a large complex of proteins of both viral and host origin and viral RNA. Some virus factories have been shown to align with and traffic along microfilaments. In addition to viral RNA replication, the factories may be involved in other processes such as viral RNA translation and cell-to-cell virus transport. Confining the process of RNA replication to a specific location may also prevent the activation of certain host defense functions.

The viral genome-linked protein, VPg, of potyviruses is a multifunctional protein involved in viral genome translation and replication. Previous studies have shown that both eukaryotic translation initiation factor 4E (eIF4E) and eIF4G or their respective isoforms from the eIF4F complex, which modulates the initiation of protein translation, selectively interact with VPg and are required for potyvirus infection. Here, we report the identification of two DEAD-box RNA helicase-like proteins, PpDDXL and AtRH8 from peach (Prunus persica) and Arabidopsis (Arabidopsis thaliana), respectively, both interacting with VPg. We show that AtRH8 is dispensable for plant growth and development but necessary for potyvirus infection. In potyvirus-infected Nicotiana benthamiana leaf tissues, AtRH8 colocalizes with the chloroplast-bound virus accumulation vesicles, suggesting a possible role of AtRH8 in viral genome translation and replication. Deletion analyses of AtRH8 have identified the VPg-binding region. Comparison of this region and the corresponding region of PpDDXL suggests that they are highly conserved and share the same secondary structure. Moreover, overexpression of the VPg-binding region from either AtRH8 or PpDDXL suppresses potyvirus accumulation in infected N. benthamiana leaf tissues. Taken together, these data demonstrate that AtRH8, interacting with VPg, is a host factor required for the potyvirus infection process and that both AtRH8 and PpDDXL may be manipulated for the development of genetic resistance against potyvirus infections.

The replication of positive-strand RNA viruses occurs in cytoplasmic membrane-bound virus replication complexes (VRCs). Depending on the virus, distinct cellular organelles such as the endoplasmic reticulum (ER), chloroplast, mitochondrion, endosome, and peroxisome are recruited for the formation of VRC-associated membranous structures. Previously, the 6,000-molecular-weight protein (6K) of plant potyviruses was shown to be an integral membrane protein that induces the formation of 6K-containing membranous vesicles at endoplasmic reticulum (ER) exit sites for potyvirus genome replication. Here, we present evidence that the 6K-induced vesicles predominantly target chloroplasts, where they amalgamate and induce chloroplast membrane invaginations. The vesicular transport pathway and actomyosin motility system are involved in the trafficking of the 6K vesicles from the ER to chloroplasts. Viral RNA, double-stranded RNA, and viral replicase components are concentrated at the 6K vesicles that associate with chloroplasts in infected cells, suggesting that these chloroplast-bound 6K vesicles are the site for potyvirus replication. Taken together, these results suggest that plant potyviruses sequentially recruit the ER and chloroplasts for their genome replication.

Purpose: To assess the reliability of Orbscan (Bausch & Lomb, Salt Lake City, UT) and Pentacam (Oculus, Wetzlar, Germany) central corneal thickness (CCT) and peripheral corneal thickness (PCT) measurements based on 2 methodologies., Design: Evaluation of a diagnostic technology., Participants: Thirty healthy volunteers were recruited prospectively at the Department of Ophthalmology of the Hôtel-Dieu Hospital, Paris, France., Methods: Central corneal thickness and PCT were assessed, using ultrasound pachymetry (USP) as the gold standard. Two methodologies were used: (1) the traditional analysis of pachymetry data from 1 central and 8 peripheral reference positions on the cornea, and (2) a 3-dimensional (3-D) analysis based on average corneal pachymetry maps constructed for each system (Orbscan, Pentacam, and USP), each operator (operators 1 and 2), and each visit (visits A and B)., Main Outcome Measures: Repeatability, intersystem reproducibility, interoperator reproducibility, reproducibility over time, and accuracy of Orbscan and Pentacam CCT and PCT measurements. Distribution and statistical significance of the differences between 3-D average maps., Results: Repeatability (Orbscan intraclass correlation coefficients [ICCs], 0.967-0.992; Pentacam ICCs, 0.986-0.997), interoperator reproducibility, and reproducibility over time (ICCs, 0.976-0.997) were excellent to almost perfect for both systems. Intersystem agreement was almost perfect for CCT (ICC, 0.980), but less strong for PCT (ICCs, 0.928-0.979). Despite a good to excellent agreement between the optical systems and USP (ICCs, 0.608-0.958), USP CCT readings were thicker (mean difference, up to 15.2 microm; P<0.05), and USP PCT readings were thinner (P<0.05). Orbscan and Pentacam average maps allowed comprehensive interpretation of differences between populations according to the magnitude, distribution, and statistical significance, minimizing the risk of giving excessive weight to few data measured at specific locations on the cornea., Conclusions: Both methodologies showed that Orbscan and Pentacam CCT readings are interchangeable, whereas caution should be used for PCT readings. Interchangeability with USP measurements also was shown to be limited. The high repeatability, interoperator reproducibility, reproducibility over time, the extent of the information generated by a single capture, and the noncontact nature of the Orbscan and Pentacam all suggest that optical systems eventually may replace USP as the gold standard for corneal pachymetry.

Nicotiana benthamiana plants were agroinoculated with an infectious cDNA clone of Turnip mosaic virus (TuMV) that was engineered to express a fluorescent protein (green fluorescent protein [GFP] or mCherry) fused to the viral 6K2 protein known to induce vesicle formation. Cytoplasmic fluorescent discrete protein structures were observed in infected cells, corresponding to the vesicles containing the viral RNA replication complex. The vesicles were motile and aligned with microfilaments. Intracellular movement of the vesicles was inhibited when cells were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. It was also observed that viral accumulation in the presence of this drug was reduced. These data indicate that microfilaments are used for vesicle movement and are necessary for virus production. Biogenesis of the vesicles was further investigated by infecting cells with two recombinant TuMV strains: one expressed 6K2GFP and the other expressed 6K2mCherry. Green- and red-only vesicles were observed within the same cell, suggesting that each vesicle originated from a single viral genome. There were also vesicles that exhibited sectors of green, red, or yellow fluorescence, an indication that fusion among individual vesicles is possible. Protoplasts derived from TuMV-infected N. benthamiana leaves were isolated. Using immunofluorescence staining and confocal microscopy, viral RNA synthesis sites were visualized as punctate structures distributed throughout the cytoplasm. The viral proteins VPg-Pro, RNA-dependent RNA polymerase, and cytoplasmic inclusion protein (helicase) and host translation factors were found to be associated with these structures. A single-genome origin and presence of protein synthetic machinery components suggest that translation of viral RNA is taking place within the vesicle.

The poly(A)-binding protein (PABP) is an important translation initiation factor that binds to the polyadenylated 3' end of mRNA. We have previously shown that PABP2 interacts with the RNA-dependent RNA polymerase (RdRp) and VPg-Pro of turnip mosaic virus (TuMV) within virus-induced vesicles. At least eight PABP isoforms are produced in Arabidopsis thaliana, three of which (PABP2, PABP4 and PABP8) are highly and broadly expressed and probably constitute the bulk of PABP required for cellular functions. Upon TuMV infection, an increase in protein and mRNA expression from PAB2, PAB4 and PAB8 genes was recorded. In vitro binding assays revealed that RdRp and the viral genome-linked protein (VPg-Pro) interact preferentially with PABP2 but are also capable of interaction with one or both of the other class II PABPs (i.e. PABP4 and PABP8). To assess whether PABP is required for potyvirus replication, A. thaliana single and double pab knockouts were isolated and inoculated with TuMV. All lines showed susceptibility to TuMV. However, when precise monitoring of viral RNA accumulation was performed, it was found to be reduced by 2.2- and 3.5-fold in pab2 pab4 and pab2 pab8 mutants, respectively, when compared with wild-type plants. PABP levels were most significantly reduced in the membrane-associated fraction in both of these mutants. TuMV mRNA levels thus correlated with cellular PABP concentrations in these A. thaliana knockout lines. These data provide further support for a role of PABP in potyvirus replication.

Eukaryotic elongation factor 1-alpha (eEF1A) was identified as an interactor of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp) and VPg-protease (VPg-Pro) using tandem affinity purification and/or in vitro assays. Subcellular fractionation experiments revealed that the level of eEF1A substantially increased in membrane fractions upon TuMV infection. Replication of TuMV occurs in cytoplasmic membrane vesicles, which are induced by 6K-VPg-Pro. Confocal microscopy indicated that eEF1A was included in these vesicles. To confirm that eEF1A was found in replication vesicles, we constructed an infectious recombinant TuMV that contains an additional copy of the 6K protein fused to the green fluorescent protein (GFP). In cells infected with this recombinant TuMV, fluorescence emitted by 6KGFP was associated with cytoplasmic membrane vesicles that contained VPg-Pro, the eukaryotic initiation factor (iso) 4E, the poly(A)-binding protein, the heat shock cognate 70-3 protein, and eEF1A. These results suggest that TuMV-induced membrane vesicles host at least three plant translation factors in addition to the viral replication proteins.

Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions.

Poly(A) binding protein 2 (PABP2) of Arabidopsis thaliana was previously shown to interact with VPg-Pro of turnip mosaic virus (TuMV) and may consequently play an important role during infection. Subcellular fractionation experiments revealed that PABP2 was predominantly a cytoplasmic soluble protein in healthy plants. However, in TuMV-infected plants, a subpopulation of PABP2 was membrane associated or was localized in the nucleus. Confocal microscopy experiments indicated that PABP2 was partially retargeted to the nucleolus in the presence of TuMV VPg-Pro. In addition, the membrane association of PABP2 during TuMV infection resulted from the internalization of the host protein in 6K-VPg-Pro-induced vesicles, as shown by a combination of confocal microscopy and sucrose gradient fractionation experiments. This redistribution of an important translation initiation factor to the nucleolus and to membrane structure likely underlies two important processes of the TuMV replication cycle.

Purpose: To evaluate consequences of misalignment during corneal topography., Setting: Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada., Methods: Twenty-nine healthy subjects were enrolled in a study of 2 types of misalignment during Orbscan topography acquisition. The first was rotation, which was defined as angular deviation of the subject's visual axis away from the central fixation target, and the second was translation, defined as off-center displacement of the joined half slits on the computer screen during alignment by the technician. The effect of vertical and horizontal misalignments, by rotation or translation, was assessed. Study parameters included simulated keratometries (SimKs), mean corneal power (mean power), and corneal apex (R) and the asphericity factor (Q) values. Inferior-superior (I-S) and temporal-nasal (T-N) indices were calculated. Differences in parameter values between aligned and misaligned positions were studied. A mathematical model was also developed to simulate rotational and translational misalignment and consolidate clinical experimentation., Results: The mean SimK values were slightly but significantly affected by rotation. The I-S value increased significantly with downward rotation and decreased with upward rotation of the eye, while T-N values increased significantly with temporal rotation and decreased with nasal rotation. The R and Q values increased with rotation. Overall, the topography parameters were minimally affected by translation. Comparable results were obtained with the theoretical model., Conclusions: Rotational misalignment of the eye during acquisition influenced topography more than the translational misalignment during focusing by the technician. Rotational misalignment induced corneal asymmetry on elevation and curvature maps as well as pseudokeratoconus patterns.

Optimized plant-microbe bioremediation processes in which the plant initiates the metabolism of xenobiotics and releases the metabolites in the rhizosphere to be further degraded by the rhizobacteria is a promising alternative to restore contaminated sites in situ. However, such processes require that plants produce the metabolites that bacteria can readily oxidize. The biphenyl dioxygenase is the first enzyme of the bacterial catabolic pathway involved in the degradation of polychlorinated biphenyls. This enzyme consists of three components: the two sub-unit oxygenase (BphAE) containing a Rieske-type iron-sulfur cluster and a mononuclear iron center, the Rieske-type ferredoxin (BphF), and the FAD-containing ferredoxin reductase (BphG). In this work, based on analyses with Nicotiana benthamiana plants transiently expressing the biphenyl dioxygenase genes from Burkholderia xenovorans LB400 and transgenic Nicotiana tabacum plants transformed with each of these four genes, we have shown that each of the three biphenyl dioxygenase components can be produced individually as active protein in tobacco plants. Therefore, when BphAE, BphF, and BphG purified from plant were used to catalyze the oxygenation of 4-chlorobiphenyl, detectable amounts of 2,3-dihydro-2, 3-dihydroxy-4'-chlorobiphenyl were produced. This suggests that creating transgenic plants expressing simultaneously all four genes required to produce active biphenyl dioxygenase is feasible.

We developed an inactivated DNA replicon of Turnip Mosaic Virus (TuMV), which was reactivated by a recombination event based on the Cre-loxP system. Viral replication was prevented by the insertion of a translation terminator sequence flanked by two loxP sites at the junction of the P1-HCPro-coding genes. In vitro recombination was tested with purified Cre, which excised the floxed sequence from the TuMV DNA, leaving a single loxP site in the reactivated viral genome, and restored the open reading frame of the replicon. Arabidopsis thaliana plants were made transgenic for the inactivated TuMV replicon. Removal of the translation terminator sequence was achieved by the controlled expression of Cre. Delivery of the Cre recombinase to the transgenic plants was obtained by three methods: agroinfiltration, PVX-based production, or transgenic chemical-inducible expression. In each case, reactivation of TuMV replication was observed.

Wheat is the most widely adapted crop to abiotic stresses and considered an excellent system to study stress tolerance in spite of its genetic complexity. Recent studies indicated that several hundred genes are either up- or down-regulated in response to stress treatment. To elucidate the function of some of these genes, an interactome of proteins associated with abiotic stress response and development in wheat was generated using the yeast two-hybrid GAL4 system and specific protein interaction assays. The interactome is comprised of 73 proteins, generating 97 interactions pairs. Twenty-one interactions were confirmed by bimolecular fluorescent complementation in Nicotiana benthamiana. A confidence-scoring system was elaborated to evaluate the significance of the interactions. The main feature of this interactome is that almost all bait proteins along with their interactors were interconnected, creating a spider web-like structure. The interactome revealed also the presence of a "cluster of proteins involved in flowering control" in three- and four-protein interaction loops. This network provides a novel insight into the complex relationships among transcription factors known to play central roles in vernalization, flower initiation and abscisic acid signaling, as well as associations that tie abiotic stress with other regulatory and signaling proteins. This analysis provides useful information in elucidating the molecular mechanism associated with abiotic stress response in plants.

Purpose: A methodology is proposed to build population-based average three dimensional (3-D) atlases or standards of the human cornea based on topographic data, along with variation maps. Also, methodologies for comparing populations or screening populations, based on these atlases are proposed., Methods: Topographies (Orbscan II; Bausch & Lomb, Rochester, NY) of 516 normal subjects were used. Methodology for the construction of a corneal atlas consisted of (1) data acquisition from both anterior and posterior corneal surfaces in the format of a 101 x 101 grid of z elevations evenly spaced (every 0.1 mm) along the x and y axes; (2) spatial normalization of the topographies on a unique average best-fit sphere to reduce the large variability in size and spatial location between corneas; (3) generation of the average 3-D model; and (4) statistics maps including average, median, and SD for each point of the grid., Results: To demonstrate the informative potential of this methodology, examples of atlases were generated. Numerical corneal atlases allow (1) characterization of a population, (2) comparison of two or more populations, (3) comparison of an individual with a reference population, and (4) screening of a population for the detection of specific corneal shape abnormalities, such as keratoconus or previous refractive surgery., Conclusions: The concept of a 3-D corneal atlas was developed. The proposed technique was meant to be simple, accurate, reliable, and robust and can be extended easily to any type of topographer capable of providing tridimensional corneal maps.

The RNA genome of Turnip mosaic virus is covalently linked at its 5' end to a viral protein known as VPg. This protein binds to the translation eukaryotic initiation factor iso 4E [eIF(iso)4E]. This interaction has been shown to be important for virus infection, although its exact biological function(s) has not been elucidated. In this study, we investigated the subcellular site of the VPg-eIF(iso)4E interaction using bimolecular fluorescence complementation (BiFC). As a first step, eIF(iso)4E, 6K-VPg-Pro, and VPg-Pro were expressed as full-length green fluorescent protein (GFP) fusions in Nicotiana benthamiana, and their subcellular localizations were visualized by confocal microscopy. eIF(iso)4E was predominantly associated with the endoplasmic reticulum (ER), and VPg-Pro was observed in the nucleus and possibly the nucleolus, while 6K-VPg-Pro-GFP induced the formation of cytoplasmic vesicles budding from the ER. In BiFC experiments, reconstituted green fluorescence was observed throughout the nucleus, with a preferential accumulation in subnuclear structures when the GFP split fragments were fused to VPg-Pro and eIF(iso)4E. On the other hand, the interaction of 6K-VPg-Pro with eIF(iso)4E was observed in cytoplasmic vesicles embedded in the ER. These data suggest that the association of VPg with the translation factor might be needed for two different functions, depending of the VPg precursor involved in the interaction. VPg-Pro interaction with eIF(iso)4E may be involved in perturbing normal cellular functions, while 6K-VPg-Pro interaction with the translation factor may be needed for viral RNA translation and/or replication.

Purpose: To determine whether donor eyes had previous refractive surgery using Orbscan (Bausch & Lomb Surgical) corneal topography., Setting: Lions Eye Bank of Oregon, Portland, Oregon, USA, and Maisonneuve-Rosemont, Hospital, Montreal, Quebec, Canada., Methods: Orbscan corneal topographies of 50 donor eyes from the Lions Eye Bank of Oregon were obtained; 10 eyes had previous refractive surgery (6 laser in situ keratomileusis, 2 photorefractive keratectomy, 2 radial keratotomy) to correct myopia, and 40 had not had surgery. Algorithms based on corneal anterior and posterior elevations and anterior tangential curvature were developed: The difference in curvature (DC) was based on the difference in the mean anterior tangential curvature between central and midperipheral areas; difference in elevation (DE) represented the difference between the anterior and posterior central elevations. Receiver-operating characteristic (ROC) curves for each algorithm were obtained, and sensitivity values at fixed specificities were calculated., Results: The mean area under the ROC curve, which corresponds to the probability of correctly identifying the presence of a previous refractive surgery, was 0.853 +/- 0.079 (SE) for DC and 0.933 +/- 0.057 for DE. The DC algorithm resulted in a sensitivity of 80% for a specificity of 87.5%, and DE yielded a sensitivity of 90% for a specificity of 92.5%. There was a strong correlation between the value of the DE and DC algorithms and the amount of previous refractive surgery (DC: r = 0.84, P = .008; DE: r = 0.76, P = .028)., Conclusion: The results led to a proposed criteria-based system using Orbscan corneal topography to screen eye-bank eyes for previous refractive surgery.

Unlike other positive-stranded RNA viruses that use either a 5'-cap structure or an internal ribosome entry site to direct translation of their messenger RNA, calicivirus translation is dependent on the presence of a protein covalently linked to the 5' end of the viral genome (VPg). We have shown a direct interaction of the calicivirus VPg with the cap-binding protein eIF 4 E. This interaction is required for calicivirus mRNA translation, as sequestration of eIF 4 E by 4 E-BP 1 inhibits translation. Functional analysis has shown that VPg does not interfere with the interaction between eIF 4 E and the cap structure or 4 E-BP 1, suggesting that VPg binds to eIF 4 E at a different site from both cap and 4 E-BP 1. This work lends support to the idea that calicivirus VPg acts as a novel 'cap substitute' during initiation of translation on virus mRNA.

With the aim of developing a biotechnological tool for the production of foreign proteins in plants, we first engineered an infectious turnip mosaic virus (TuMV) cDNA that contained the jellyfish green fluorescent protein (GFP) gene or the bacterial beta-glucuronidase (GUS) gene (uidA). Two insertion sites were assessed, either between P1 and HCPro cistrons or Pol and CP cistrons. In each construct, the junctions flanking the inserted gene coded for P1 and/or VPg-Pro cleavage recognition site sequences, to produce free GUS or GFP. After transfection by particle bombardment on Brassica perviridis, characteristic symptoms for TuMV infection appeared and Western blot analyses showed that GFP and GUS had been excised from the viral polyprotein. No significant differences in expression level were noticed between the two insertion sites. By RT-PCR, gfp was found to be stable over 30 days post-transfection (dpt) while uidA was gradually lost at 15 dpt. We also created two constructs containing either gene at each insertion sites on the same molecule. Attenuated systemic symptoms were observed after particle bombardment on B. perviridis and Western blot analyses showed that both foreign proteins were produced. Also, the same stability/instability as for the single-gene constructs were observed. These results indicate that it is possible to produce at least two foreign proteins simultaneously in a TuMV-based vector.

The initiation of the reproductive phase in winter cereals is delayed during winter until favorable growth conditions resume in the spring. This delay is modulated by low temperature through the process of vernalization. The molecular and genetic bases of the interaction between environmental factors and the floral transition in these species are still unknown. However, the recent identification of the wheat (Triticum aestivum L.) TaVRT-1 gene provides an opportunity to decipher the molecular basis of the flowering-time regulation in cereals. Here, we describe the characterization of another gene, named TaVRT-2, possibly involved in the flowering pathway in wheat. Molecular and phylogenetic analyses indicate that the gene encodes a member of the MADS-box transcription factor family that belongs to a clade responsible for flowering repression in several species. Expression profiling of TaVRT-2 in near-isogenic lines and different genotypes with natural variation in their response to vernalization and photoperiod showed a strong relationship with floral transition. Its expression is up-regulated in the winter genotypes during the vegetative phase and in photoperiod-sensitive genotypes during short days, and is repressed by vernalization to a level that allows the transition to the reproductive phase. Protein-protein interaction studies revealed that TaVRT-2 interacts with proteins encoded by two important vernalization genes (TaVRT-1/VRN-1 and VRN-2) in wheat. These results support the hypothesis that TaVRT-2 is a putative repressor of the floral transition in wheat.

Positive-sense single-stranded RNA viruses have developed strategies to exploit cellular resources at the expense of host mRNAs. The genomes of these viruses display a variety of structures at their 5' and 3' ends that differentiate them from cellular mRNAs. Despite this structural diversity, viral RNAs are still circularized by juxtaposition of their 5' and 3' ends, similar to the process used by cellular mRNAs. Also reminiscent of the mechanisms used by host mRNAs, translation of viral RNAs involves the recruitment of translation initiation factors. However, the roles played by these factors likely differ from those played by cellular mRNAs. In keeping with the general parsimony typical of RNA viruses, these host factors also participate in viral RNA replication. However, the dual use of host factors requires that viral RNA template utilization be regulated to avoid conflict between replication and translation. The molecular composition of the large ribonucleoprotein complexes that form the viral RNA replication and translation machineries likely evolves over the course of infection to allow for switching template use from translation to replication.

Purpose: To develop a screening tool based on corneal topography to detect previous myopic and hyperopic laser in situ keratomileusis (LASIK)., Methods: Clinical data from three private clinics were analyzed in a university setting. A total of 476 topographies (Orbscan II) were randomly selected (1 topography per patient): 338 from unoperated corneas, 81 from corneas that had LASIK to correct myopia, and 57 from corneas that had LASIK to correct hyperopia. The LASIK procedures were performed using a Technolas excimer laser (217C or 217Z) and a Hansatome microkeratome. The first set of algorithms (VESm and VESh) calculated the volume summation between the anterior corneal surface and the best-fit sphere in the central and mid-peripheral regions. The second set of algorithms (DCm and DCh) calculated the mean anterior tangential curvature difference between the central and mid-peripheral regions., Results: The features VESm and DCm for the screening of a myopic LASIK yield, respectively, sensitivity rates of 92.7% and 95.1% and specificity rates of 94.9% and 96.5%. The features VESh and DCh for the screening of a hyperopic LASIK gave sensitivity rates of 93.1% for both and specificity rates of 94.8% and 97.1%. The performance of those features was superior to clinical assessment using topography color maps., Conclusions: Criteria based on Orbscan II corneal topography are proposed for the detection of previous hyperopic and myopic LASIK performed with a Technolas excimer laser (version 217C or 217Z).

The viral protein linked to the genome (VPg) of Turnip mosaic virus (TuMV) interacts in vitro with the translation eukaryotic initiation factor (eIF) 4E. In the present study, we investigated the consequence of TuMV infection on eIF4E expression. Two isomers are present in plants, namely eIF4E and eIF(iso)4E. Expression of the latter was detected in both TuMV-infected and mock-inoculated Brassica perviridis plants, but expression of eIF4E was found only in infected plants. Membranes from TuMV-infected or mock-inoculated tissues were separated by sucrose gradient centrifugation and fractions were collected. Immunoblot analyses showed that 6K(2)-VPg-Pro/VPg-Pro polyproteins were associated with endoplasmic reticulum membranes and were the viral forms likely to interact with eIF(iso)4E and eIF4E. In planta interaction between 6K(2)-VPg-Pro/VPg-Pro and eIF(iso)4E/eIF4E was confirmed by co-purification by metal chelation chromatography. The poly(A)-binding protein (PABP) was also found to co-purify with VPg-Pro. Direct interaction between VPg-Pro and PABP was shown by an ELISA-based binding assay. These experiments suggest that a multi-protein complex may form around VPg-Pro of TuMV.

Eukaryotic initiation factor eIF(iso)4E binds to the cap structure of mRNAs leading to assembly of the translation complex. This factor also interacts with the potyvirus VPg and this interaction has been correlated with virus infectivity. In this study, we show an interaction between eIF(iso)4E and the proteinase (Pro) of a nepovirus (Tomato ringspot virus; ToRSV) in vitro. The ToRSV VPg did not interact with eIF(iso)4E although its presence on the VPg-Pro precursor increased the binding affinity of Pro for the initiation factor. A major determinant of the interaction was mapped to the first 93 residues of Pro. Formation of the complex was inhibited by addition of m(7)GTP (a cap analogue), suggesting that Pro-containing molecules compete with cellular mRNAs for eIF(iso)4E binding. The possible implications of this interaction for translation and/or replication of the virus genome are discussed.

The interaction between the viral protein linked to the genome (VPg) of turnip mosaic potyvirus (TuMV) and the translation eukaryotic initiation factor eIF(iso)4E of Arabidopsis thaliana has previously been reported. eIF(iso)4E binds the cap structure (m(7)GpppN, where N is any nucleotide) of mRNAs and has an important role in the regulation in the initiation of translation. In the present study, it was shown that not only did VPg bind eIF(iso)4E but it also interacted with the eIF4E isomer of A. thaliana as well as with eIF(iso)4E of Triticum aestivum (wheat). The interaction domain on VPg was mapped to a stretch of 35 amino acids, and substitution of an aspartic acid residue found within this region completely abolished the interaction. The cap analogue m(7)GTP, but not GTP, inhibited VPg-eIF(iso)4E complex formation, suggesting that VPg and cellular mRNAs compete for eIF(iso)4E binding. The biological significance of this interaction was investigated. Brassica perviridis plants were infected with a TuMV infectious cDNA (p35Tunos) and p35TuD77N, a mutant which contained the aspartic acid substitution in the VPg domain that abolished the interaction with eIF(iso)4E. After 20 days, plants bombarded with p35Tunos showed viral symptoms, while plants bombarded with p35TuD77N remained symptomless. These results suggest that VPg-eIF(iso)4E interaction is a critical element for virus production.

ABSTRACT Eight turnip mosaic potyvirus (TuMV) isolates from the Campania region of Italy were characterized. Experiments based on host range and symptomatology indicated that the isolates were biologically different. In addition, the isolates, with the exception of ITA1 and ITA3, were distinguished from each other by using a combination of monoclonal antibodies recognizing the coat protein. Single-strand conformation polymorphism (SSCP) analysis of the coat protein gene revealed that each isolate produced a specific SSCP profile, except for isolates ITA1 and ITA3. This study indicates that (i) even in a small geographical region, there is a great deal of variation in TuMV isolates; (ii) the use of a set of four differential hosts does not always specify the same pathotype in different environments; (iii) the TuMV isolates with the same pathotype on Brassica napus test lines can still differ in host range, symptoms, serology, and SSCP; and (iv) there was perfect correlation between the panel of antibodies and SSCP in differentiating among the isolates; ITA1 and ITA3 were indistinguishable by either assay.

Sequencing of complementary DNAs prepared from various coronaviruses has revealed open reading frames encoding putative proteins that are yet to be characterized and are so far only described as nonstructural (ns). As a first step in the elucidation of its function, we characterized the expression and immunogenicity of the ns4b gene product from strain 229E of human coronavirus (HCV-229E), a respiratory virus with a neurotropic potential. The gene was cloned and expressed in bacteria. A fusion protein of ns4b with maltose-binding protein was injected into rabbits to generate specific antibodies that were used to demonstrate the expression of ns4b in HCV-229E-infected cells using flow cytometry. Given a previously reported contiguous five amino acid shared region between ns4b and myelin basic protein, a purified recombinant histidine-tagged ns4b protein and (or) human myelin basic protein were injected into mice to evaluate whether myelin-viral protein cross-reactive antibody responses could be generated. Each immunogen induced specific but not cross-reactive antibodies. We conclude that ns4b is expressed in infected cells and is immunogenic, although this does not involve amino acids shared with a self protein, at least in the experimental conditions used.

Experimental data are provided for the presence of a plant protein that interacts with the capsid protein (CP) of turnip mosaic potyvirus (TuMV). The receptor-like protein was identified by exploiting the molecular mimicry potential of anti-idiotypic antibodies. A single-chain Fv molecule derived from the monoclonal antibody 7A (Mab-7A), which recognizes the CP of TuMV, was produced in Escherichia coli and the recombinant protein was used to raise rabbit antibodies. The immune serum reacted with Mab-7A but not with a monoclonal antibody of the same isotype, indicating that anti-idiotypic antibodies were produced. These anti-idiotypic antibodies recognized a 37 kDa protein from Lactuca sativa. Complex formation between the anti-idiotypic antibodies and the plant protein was inhibited by the CP of TuMV which indicates that the plant protein interacts with the viral protein. The 37 kDa protein was localized in chloroplasts and was detected in other plant species.

The yeast LexA interaction trap was used to screen a cDNA library from Arabidopsis thaliana in order to identify proteins that interact with the viral protein genome linked (VPg)-proteinase of turnip mosaic potyvirus. The screen allowed the isolation of four candidate cDNA clones. Clones pHC4, pHC21, and pHC40 were partially sequenced but no homologies to known proteins were found. However, the amino acid sequence deduced from the complete nucleotide sequence of pSW56 revealed that it was the eukaryotic initiation factor (iso) 4E [eIF(iso)4E]. Deletion analysis indicated that the VPg domain was involved in the interaction with the plant protein. Interaction between the viral protein and the cellular protein was confirmed by ELISA-based binding experiments. eIF(iso)4E plays an essential role in the initiation of the translation of capped mRNAs and its association with VPg would point to a role of the viral protein in the translation of the virus.

We identified subgroup specific protective epitopes represented by the amino acid regions 174-187 and 171-187 of the G glycoproteins from respiratory syncytial virus (RSV), subgroups A and B. Mice immunized with coupled synthetic peptides corresponding to either the region 174-187 containing a Cys186-->Ser substitution or to the native region 171-187 were completely resistant to RSV infection but only to the respective virus. The protective activities of the peptides 174-187 were dependent on the Cys186-->Ser substitution. In addition, a recombinant protein representing the region 125-203 of the A subgroup G glycoprotein expressed in Escherichia coli was capable without further treatment to completely protect animals against RSV subgroup A infection. We show that the combinations of cysteinyl residues (positions 173, 176, 182, and 186) retained within either synthetic peptides or the recombinant protein G125-203 greatly influenced their protective activities. This indicates that the region 171-187 is essential for the protection conferred by the G125-203 protein. Furthermore, our results strongly suggest that the peptides' and recombinant protein's potencies are a function of a loop-like structure which is stabilized by intramolecular disulfide linkages between Cys176-Cys182 and Cys173-Cys186. This is further supported by the observation that chemical blocking of the sulfidryl groups in synthetic peptides completely eliminated their protective activity.