Dynamin-related protein 2 interacts with the membrane-associated methyltransferase domain of plantago asiatica mosaic virus replicase and promotes viral replication.

Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus, in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana, AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the interaction of NbDRP2 with the MET domain in PlAMV replication.

Structural basis for proteolytic processing of Aspergillus sojae α-glucosidase L with strong transglucosylation activity.

An α-glucosidase from Aspergillus sojae, AsojAgdL, exhibits strong transglucosylation activity to produce α-1,6-glucosidic linkages. The most remarkable structural feature of AsojAgdL is that residues 457-560 of AsojAgdL (designated the NC sequence) is not conserved in other glycoside hydrolase family 31 enzymes, and part of this NC sequence is proteolytically cleaved during its maturation. In this study, the enzyme was expressed in Pichia pastoris, and electrophoretic analysis indicated that the recombinant enzyme, rAsojAgdL, consisted of two polypeptide chains, as observed in the case of the enzyme produced in an Aspergillus strain. The crystal structure of rAsojAgdL was determined in complex with the substrate analog trehalose. Electron density corresponding to residues 496-515 of the NC sequence was not seen, and there were no α-helices or ß-strands except for a short α-helix in the structures of residues 457-495 and residues 516-560, both of which belong to the NC sequence. The residues 457-495 and the residues 516-560 both formed extra components of the catalytic domain. The residues 457-495 constituted the entrance of the catalytic pocket of rAsojAgdL, and Gly467, Asp468, Pro469, and Pro470 in the NC sequence were located within 4 Å of Trp400, a key residue involved in binding of the substrate. The results suggest that the proteolytic processing of the NC sequence is related to the formation of the catalytic pocket of AsojAgdL.

Acibenzolar-S-methyl-mediated restriction of loading of plantago asiatica mosaic virus into vascular tissues of Nicotiana benthamiana.

Long-distance movement via vascular tissues is an essential step for systemic infection by plant viruses. We previously reported that pre-treatment of Nicotiana benthamiana with acibenzolar-S-methyl (ASM) both suppressed the accumulation of plantago asiatica mosaic virus (PlAMV) in inoculated leaves and delayed the long-distance movement to uninoculated upper leaves. These two effects occurred independently of each other. However, it remained unclear where and when the viral long-distance movement is inhibited upon ASM treatment. In this study, we found that ASM treatment restricted the loading of GFP-expressing PlAMV (PlAMV-GFP) into vascular tissues in the inoculated leaves. This led to delays in viral translocation to the petiole and the main stem, and to untreated upper leaves. We used cryohistological fluorescence imaging to show that ASM treatment affected the viral localization and reduced its accumulation in the phloem, xylem, and mesophyll tissues. A stem girdling experiment, which blocked viral movement downward through phloem tissues, demonstrated that ASM treatment could inhibit viral systemic infection to upper leaves, which occurred even with viral downward movement restricted. Taken together, our results showed that ASM treatment affects the loading of PlAMV-GFP into the vascular system in the inoculated leaf, and that this plays a key role in the ASM-mediated delay of viral long-distance movement.

Identification of a Proline-Kinked Amphipathic α-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation.

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

Cooperative roles of introns 1 and 2 of tobacco resistance gene N in enhanced N transcript expression and antiviral defense responses.

The tobacco virus resistance gene N contains four introns. Transient expression of transcripts from an N transgene containing these introns and driven by the native promoter in the presence of the elicitor of tobacco mosaic virus resulted in its increased expression. The requirement of the native promoter, the elicitor, or the individual introns for enhanced expression of N has not been fully studied. Here, we determined that 35S promoter-driven N transcript expression could be enhanced in the presence of the four introns regardless of the co-expression of the virus elicitor in tobacco. Function analyses using a series of N transgenes with different combination of introns revealed that the presence of intron 1 more so than intron 2 allowed higher accumulation of premature and mature N transcripts; however, both introns were important for not only enhanced gene expression but also for induction of cell death in tobacco and induced local resistance to spread of virus in Nicotiana benthamiana. Our findings indicate that introns 1 and 2 cooperatively contribute to N expression and virus resistance.

Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by Tobamovirus Infection and Transient Expression of Viral Replication and Movement Proteins.

Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexvirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by tomato mosaic virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamovirus movement.

Identification of a movement protein of Mirafiori lettuce big-vein ophiovirus.

Mirafiori lettuce big-vein virus (MiLBVV) is a member of the genus Ophiovirus, which is a segmented negative-stranded RNA virus. In microprojectile bombardment experiments to identify a movement protein (MP) gene of ophioviruses that can trans-complement intercellular movement of an MP-deficient heterologous virus, a plasmid containing an infectious clone of a tomato mosaic virus (ToMV) derivative expressing the GFP was co-bombarded with plasmids containing one of three genes from MiLBVV RNAs 1, 2 and 4 onto Nicotiana benthamiana. Intercellular movement of the movement-defective ToMV was restored by co-expression of the 55 kDa protein gene, but not with the two other genes. Transient expression in epidermal cells of N. benthamiana and onion showed that the 55 kDa protein with GFP was localized on the plasmodesmata. The 55 kDa protein encoded in the MiLBVV RNA2 can function as an MP of the virus. This report is the first to describe an ophiovirus MP.

The movement protein encoded by gene 3 of rice transitory yellowing virus is associated with virus particles.

Gene 3 in the genomes of several plant-infecting rhabdoviruses, including rice transitory yellowing virus (RTYV), has been postulated to encode a cell-to-cell movement protein (MP). Trans-complementation experiments using a movement-defective tomato mosaic virus and the P3 protein of RTYV, encoded by gene 3, facilitated intercellular transport of the mutant virus. In transient-expression experiments with the GFP-fused P3 protein in epidermal leaf cells of Nicotiana benthamiana, the P3 protein was associated with the nucleus and plasmodesmata. Immunogold-labelling studies of thin sections of RTYV-infected rice plants using an antiserum against Escherichia coli-expressed His(6)-tagged P3 protein indicated that the P3 protein was located in cell walls and on virus particles. In Western blots using antisera against E. coli-expressed P3 protein and purified RTYV, the P3 protein was detected in purified RTYV, whilst antiserum against purified RTYV reacted with the E. coli-expressed P3 protein. After immunogold labelling of crude sap from RTYV-infected rice leaves, the P3 protein, as well as the N protein, was detected on the ribonucleocapsid core that emerged from partially disrupted virus particles. These results provide evidence that the P3 protein of RTYV, which functions as a viral MP, is a viral structural protein and seems to be associated with the ribonucleocapsid core of virus particles.

Differing requirements for actin and myosin by plant viruses for sustained intercellular movement.

The actin cytoskeleton has been implicated in the intra- and intercellular movement of a growing number of plant and animal viruses. However, the range of viruses influenced by actin for movement and the mechanism of this transport are poorly understood. Here we determine the importance of microfilaments and myosins for the sustained intercellular movement of a group of RNA-based plant viruses. We demonstrate that the intercellular movement of viruses from different genera [tobacco mosaic virus (TMV), potato virus X (PVX), tomato bushy stunt virus (TBSV)], is inhibited by disruption of microfilaments. Surprisingly, turnip vein-clearing virus (TVCV), a virus from the same genus as TMV, did not require intact microfilaments for normal spread. To investigate the molecular basis for this difference we compared the subcellular location of GFP fusions to the 126-kDa protein and the homologous 125-kDa protein from TMV and TVCV, respectively. The 126-kDa protein formed numerous large cytoplasmic inclusions associated with microfilaments, whereas the 125-kDa protein formed few small possible inclusions, none associated with microfilaments. The dependence of TMV, PVX, and TBSV on intact microfilaments for intercellular movement led us to investigate the role of myosin motors in this process. Virus-induced gene silencing of the Nicotiana benthamiana myosin XI-2 gene, but not three other myosins, inhibited only TMV movement. These results indicate that RNA viruses have evolved differently in their requirements for microfilaments and the associated myosin motors, in a manner not correlated with predicted phylogeny.

Over-expression of putative transcriptional coactivator KELP interferes with Tomato mosaic virus cell-to-cell movement.

Tomato mosaic virus (ToMV) encodes a movement protein (MP) that is necessary for virus cell-to-cell movement. We have demonstrated previously that KELP, a putative transcriptional coactivator of Arabidopsis thaliana, and its orthologue from Brassica campestris can bind to ToMV MP in vitro. In this study, we examined the effects of the transient over-expression of KELP on ToMV infection and the intracellular localization of MP in Nicotiana benthamiana, an experimental host of the virus. In co-bombardment experiments, the over-expression of KELP inhibited virus cell-to-cell movement. The N-terminal half of KELP (KELPdC), which had been shown to bind to MP, was sufficient for inhibition. Furthermore, the over-expression of KELP and KELPdC, both of which were co-localized with ToMV MP, led to a reduction in the plasmodesmal association of MP. In the absence of MP expression, KELP was localized in the nucleus and the cytoplasm by the localization signal in its N-terminal half. It was also shown that ToMV amplified normally in protoplasts prepared from leaf tissue that expressed KELP transiently. These results indicate that over-expressed KELP interacts with MP in vivo and exerts an inhibitory effect on MP function for virus cell-to-cell movement, but not on virus amplification in individual cells.

The cysteine-histidine-rich region of the movement protein of Cucumber mosaic virus contributes to plasmodesmal targeting, zinc binding and pathogenesis.

Viral movement proteins (MPs) are central to the establishment of viral pathogenesis, and yet relatively little is understood about the structural and functional aspects of MPs or about the host factors on which they depend. Through chemical mutagenesis of transgenic Arabidopsis expressing Cucumber mosaic virus (CMV) MP fused with the green fluorescent protein, we have studied the function of a central region of the MP, defined by a number of conserved cysteine and histidine residues (Cys-His-rich region), which potentially functions as a zinc-binding domain. Transient expression of mutant MPs identified through an in planta screen for altered MP function or constructed with altered putative zinc ligands through site-directed mutagenesis showed that mutations in the Cys-His-rich region affected localization to and trafficking through plasmodesmata. In vitro zinc-binding analysis revealed that wild type (wt) CMV MP had the ability to bind zinc and that movement-defective mutants bound zinc with less affinity than wt MP. Furthermore, a correlation between the association of the MP with plasmodesmata and virus pathogenesis was shown. We discuss roles of the Cys-His region in biochemical and biological functions of the MP during virus movement.

Usefulness of per-rectal portal scintigraphy with technetium-99m pertechnetate for prognosis of primary biliary cirrhosis.

BACKGROUND/AIMS: Conventional methods predicting survival in patients with primary biliary cirrhosis are based on the results of blood tests and on clinical condition, both of which may be affected by treatment. Portal circulation can be evaluated in a relatively noninvasive manner by per-rectal portal scintigraphy. We used this method to evaluate portal hemodynamics and assess prognosis in patients with primary biliary cirrhosis. METHODOLOGY: Per-rectal portal scintigraphy with Tc-99m pertechnetate was done in 51 patients with primary biliary cirrhosis. A solution containing Tc-99m pertechnetate was instilled into the rectum, and serial scintigrams were taken while radioactivity curves for the liver and heart were recorded sequentially. The per-rectal portal shunt index was calculated from the curves. RESULTS: The shunt index was higher in patients with stage IV primary biliary cirrhosis than in those with stage I, II, or III primary biliary cirrhosis. On the basis of portal shunt index, the patients were divided into those with a shunt index of less than 18%, and those with a shunt index of 18% or more. The cumulative survival rate was lower among patients with the higher shunt index. On regression analysis, the portal shunt index was found to be significantly related to survival. CONCLUSIONS: Our results indicate that per-rectal portal scintigraphy with Tc-99m pertechnetate can be used to non-invasively evaluate the portal circulation of patients with primary biliary cirrhosis and is useful in establishing prognosis in such patients.