Tobacco Etch Virus Research Articles

Temperature induced structural transitions from native to unfolded aggregated states of tobacco etch virus protease

Tobacco etch virus protease (TEVp) is widely used to remove fusion tags from recombinant proteins because of its high and unique specificity. This work describes the conformational and the thermodynamic properties in the unfolding/refolding process of TEVp3M (three-point mutant: L56V/S135G/S219V) induced by temperature. With temperature increasing from 20 to 100°C, the CD spectra showed a transition trend from α-helix to β-sheet, and the fluorescence emission, synchronous fluorescence, ANS and RLS spectroscopy consistently revealed that the temperature-induced unfolding process behaved in a three-state manner, for there was a relatively stable intermediate state observed around 50°C. The reversibility of thermal unfolding of TEVp3M further showed that the transition from the native to the intermediate state was reversible (below 50°C), however the transition from the intermediate to the unfolded state was irreversible (above 60°C). Moreover, aggregates were observed above 60°C as revealed by SDS–PAGE, Thioflavin-T fluorescence and Congo red absorbance.

The pCri System: a vector collection for recombinant protein expression and purification.

A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5′end and multiple cloning sites at the 3′end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N- and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies.

An optimized intein-mediated protein ligation approach for the efficient cyclization of cysteine-rich proteins.

Head-to-tail backbone cyclization of proteins is a widely used approach for the improvement of protein stability. One way to obtain cyclic proteins via recombinant expression makes use of engineered Intein tags, which are self-cleaving protein domains. In this approach, pH-induced self-cleavage of the N-terminal Intein tag generates an N-terminal cysteine residue at the target protein, which then attacks in an intramolecular reaction the C-terminal thioester formed by the second C-terminal Intein tag resulting in the release of the cyclic target protein. In the current work we aimed to produce a cyclic analog of the small γ-Ec-1 domain of the wheat metallothionein, which contains six cysteine residues. During the purification process we faced several challenges, among them premature cleavage of one or the other Intein tag resulting in decreasing yields and contamination with linear species. To improve efficiency of the system we applied a number of optimizations such as the introduction of a Tobacco etch virus cleavage site and an additional poly-histidine tag. Our efforts resulted in the production of a cyclic protein in moderate yields without any contamination with linear protein species.

Replication-associated proteins encoded by Wheat dwarf virus act as RNA silencing suppressors

Wheat dwarf virus (WDV) is an economically important and widespread single-stranded DNA virus in the genus Mastrevirus, family Geminiviridae. So far, an RNA silencing suppressor in the genus Mastrevirus has not been reported. In this study, the viral replication-associated proteins of WDV, Rep and RepA proteins, were demonstrated to have RNA silencing suppressor activities when expressed in agro-infiltrated leaves of Nicotiana benthamiana line 16c, by their ability to inhibit posttranscriptional gene silencing (PTGS) of green fluorescent protein (GFP). On the basis of the analysis of GFP expression and siRNA accumulation, we concluded that both Rep and RepA proteins suppressed local silencing induced by either single-stranded GFP (ssGFP) or inverted repeat GFP (dsGFP) constructs, although their activity can be characterized as relatively weak in comparison to HC-Pro of tobacco etch virus.

Tobacco etch virus protein P1 traffics to the nucleolus and associates with the host 60S ribosomal subunits during infection.

The genus Potyvirus comprises a large group of positive-strand RNA plant viruses whose genome encodes a large polyprotein processed by three viral proteinases. P1 protein, the most amino-terminal product of the polyprotein, is an accessory factor stimulating viral genome amplification whose role during infection is not well understood. We infected plants with Tobacco etch virus (TEV; genus Potyvirus) clones in which P1 was tagged with a fluorescent protein to track its expression and subcellular localization or with an affinity tag to identify host proteins involved in complexes in which P1 also takes part during infection. Our results showed that TEV P1 exclusively accumulates in infected cells at an early stage of infection and that the protein displays a dynamic subcellular localization, trafficking in and out of the nucleus and nucleolus during infection. Inside the nucleolus, P1 particularly targets the dense granular component. Consistently, we found functional nucleolar localization and nuclear export signals in TEV P1 sequence. Our results also indicated that TEV P1 physically interacts with the host 80S cytoplasmic ribosomes and specifically binds to the 60S ribosomal subunits during infection. In vitro translation assays of reporter proteins suggested that TEV P1 stimulates protein translation, particularly when driven from the TEV internal ribosome entry site. These in vitro assays also suggested that TEV helper-component proteinase (HC-Pro) inhibits protein translation. Based on these findings, we propose that TEV P1 stimulates translation of viral proteins in infected cells. In this work, we researched the role during infection of tobacco etch virus P1 protease. P1 is the most mysterious protein of potyviruses, a relevant group of RNA viruses infecting plants. Our experiments showed that the viral P1 protein exclusively accumulates in infected cells at an early stage of infection and moves in and out of the nucleus of infected cells, particularly targeting the nucleolus. Our experiments also showed that P1 protein binds host ribosomes during infection. Based on these findings and other in vitro experiments we propose that P1 protein stimulates translation of viral proteins during infection.

His224 alters the R2 drug binding site and Phe218 influences the catalytic efficiency of the metallo-β-lactamase VIM-7.

Metallo-β-lactamases (MBLs) are the causative mechanism for resistance to β-lactams, including carbapenems, in many Gram-negative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (T(m)) of 48.5°C, compared to 55.3 °C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84, and Arg121. The tVIM-7-F218Y mutant had enhanced activity compared to wild-type tVIM-7, and a slightly higher Tm (57.1 °C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (T(m), 62.9 °C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly toward the positively charged cephalosporins like cefepime and ceftazidime.

First Report of Wild tomato mosaic virus Infecting Tobacco (Nicotiana tabacum) in China

Wild tomato mosaic virus (WTMV), a potyvirus, has been reported in Laichau, Vietnam, infecting Solanum torvum (wild tomato) in 2008 (3), and Kanchanaburi, Thailand, infecting Capsicum spp. in 2013 (KF250353). In mid-May 2013, Nicotiana tabacum showing yellowing, mosaic, and/or ringspot symptoms were found in natural tobacco fields of Nanxiong, Guangdong Province, China. Total RNA was extracted from symptomatic leaves and reverse transcribed with M4T (5'-GTTTTCCCAGTCACGAC (T)15-3') as the 3' anchoring primer (1). The cDNA was used as template in a PCR assay using primers M4: 5'-GTTTTCCCAGTCACGAC-3' and Sprimer: 5'-GGXAAYAAYAGYGGXCAZCC-3', which amplifies a region comprising part of the NIb protein gene, the entire coat protein (CP) gene and the 3' nontranslated region (UTR) of a potyvirus (1). A ~1,700-bp product was amplified from the cDNA derived from three of the five diseased plants. The product (KF639967) showed 87% and 84% nucleotide sequence identities with those of WTMV isolates KAN and Laichau, respectively. The CP deduced from the sequence of the product shared 87% and 86% nucleotide and 94% and 93% amino acid sequence identities with those of WTMV isolates KAN and Laichau, respectively. The 3'-UTR of the putative virus shared 93% and 92% nucleotide sequence identities to those of WTMV isolates KAN and Laichau, respectively. Thus, according to the molecular criteria for potyvirus species demarcation (2), the virus we identified should be an isolate of WTMV (isolate GD1). One of the diseased samples was homogenized in 0.1 mol/liter phosphate buffer (pH 7.0) and used to inoculate the potyvirus to healthy, two to four leaf-stage Capsicum annuum L., N. tabacum, and N. benthamiana. The inoculated, as well as mock-treated plants, which were inoculated only with phosphate buffer, were grown in soil under 12 h day/12 h night at 25°C. All inoculated N. tabacum and N. benthamiana plants developed yellowing and mosaic symptoms by 14 days post inoculation (dpi). For N. benthamiana, the symptom became very severe by 21 dpi and some diseased plants died prematurely. About 10% of inoculated C. annuum L. developed very mild veinal chlorosis 18 dpi. Cloning and sequencing experiments showed that all the symptomatic plants tested were WTMV positive, but Cucumber mosaic virus, Tobacco mosaic virus, and Tobacco etch virus negative. To our knowledge, this is the first report of WTMV in China. Also, it is the first report that WTMV infects Nicotiana spp. Although further experiments are needed to definitively attribute the disease observed in the field to WTMV, our results indicate that WTMV, which forms a monophyletic clade with a number of other potyviruses infecting Solanaceae species in phylogenetic analysis, is widely distributed, or is spreading in Southeast Asia. It may pose a threat to Solanaceae species cultivation in this region.

Resolution of telomere associations by TRF1 cleavage in mouse embryonic stem cells.

Telomere associations have been observed during key cellular processes such as mitosis, meiosis, and carcinogenesis and must be resolved before cell division to prevent genome instability. Here we establish that telomeric repeat-binding factor 1 (TRF1), a core component of the telomere protein complex, is a mediator of telomere associations in mammalian cells. Using live-cell imaging, we show that expression of TRF1 or yellow fluorescent protein (YFP)-TRF1 fusion protein above endogenous levels prevents proper telomere resolution during mitosis. TRF1 overexpression results in telomere anaphase bridges and aggregates containing TRF1 protein and telomeric DNA. Site-specific protein cleavage of YFP-TRF1 by tobacco etch virus protease resolves telomere aggregates, indicating that telomere associations are mediated by TRF1. This study provides novel insight into the formation and resolution of telomere associations.

First Report of Potato spindle tuber viroid Naturally Infecting Field Tomatoes in the Dominican Republic.

In recent years, viroid disease outbreaks have resulted in serious economic losses to a number of tomato growers in North America (1,2,3). At least three pospiviroids have been identified as the causal agents of tomato disease, including Potato spindle tuber viroid (PSTVd), Tomato chlorotic dwarf viroid (TCDVd), and Mexican papita viroid (MPVd). In the spring of 2013, a severe disease outbreak with virus-like symptoms (chlorosis and plant stunting) was observed in a tomato field located in the Dominican Republic, whose tomato production is generally exported to the United States in the winter months. The transplants were produced in house. The disease has reached an epidemic level with many diseased plants pulled and disposed of accordingly. Three samples collected in May of 2013 were screened by ELISA against 16 common tomato viruses (Alfalfa mosaic virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Pepino mosaic virus, Potato virus X, Potato virus Y, Tobacco etch virus, Tobacco mosaic virus, Tobacco ringspot virus, Tomato aspermy virus, Tomato bushy stunt virus, Tomato mosaic virus, Tomato ringspot virus, Tomato spotted wilt virus, Groundnut ringspot virus, and Tomato chlorotic spot virus), a virus group (Potyvirus group), three bacteria (Clavibacter michiganensis subsp. michiganensis, Pectobacterium atrosepticum, and Xanthomonas spp.), and Phytophthora spp. No positive result was observed, despite the presence of symptoms typical of a viral-like disease. Further analysis by RT-PCR using Agdia's proprietary pospiviroid group-specific primer resulted in positive reactions in all three samples. To determine which species of pospiviroid was present in these tomato samples, full-genomic products of the expected size (~360 bp) were amplified by RT-PCR using specific primers for PSTVd (4) and cloned using TOPO-TA cloning kit (Invitrogen, CA). A total of 8 to 10 clones from each isolate were selected for sequencing. Sequences from each clone were nearly identical and the predominant sequence DR13-01 was deposited in GenBank (Accession No. KF683200). BLASTn searches into the NCBI database demonstrated that isolate DR13-01 shared 97% sequence identity to PSTVd isolates identified in wild Solanum (U51895), cape gooseberry (EU862231), or pepper (AY532803), and 96% identity to the tomato-infecting PSTVd isolate from the United States (JX280944). The relatively lower genome sequence identity (96%) to the tomato-infecting PSTVd isolate in the United States (JX280944) suggests that PSTVd from the Dominican Republic was likely introduced from a different source, although the exact source that resulted in the current disease outbreak remains unknown. It may be the result of an inadvertent introduction of contaminated tomato seed lots or simply from local wild plants. Further investigation is necessary to determine the likely source and route of introduction of PSTVd identified in the current epidemic. Thus, proper control measures could be recommended for disease management. The detection of this viroid disease outbreak in the Dominican Republic represents further geographic expansion of the viroid disease in tomatoes beyond North America.

Neurogranin Alters the Structure and Calcium Binding Properties of Calmodulin

Neurogranin (Ng) is a member of the IQ motif class of calmodulin (CaM)-binding proteins, and interactions with CaM are its only known biological function. In this report we demonstrate that the binding affinity of Ng for CaM is weakened by Ca(2+) but to a lesser extent (2-3-fold) than that previously suggested from qualitative observations. We also show that Ng induced a >10-fold decrease in the affinity of Ca(2+) binding to the C-terminal domain of CaM with an associated increase in the Ca(2+) dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca(2+) binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca(2+)-saturated. Domain mapping using synthetic peptides indicated that the IQ motif of Ng is a poor mimetic of the intact protein and that the acidic sequence just N-terminal to the IQ motif plays an important role in reproducing Ng-mediated decreases in the Ca(2+) binding affinity of CaM. Using NMR, full-length Ng was shown to make contacts largely with residues in the C-domain of CaM, although contacts were also detected in residues in the N-terminal domain. Together, our results can be consolidated into a model where Ng contacts residues in the N- and C-lobes of both apo- and Ca(2+)-bound CaM and that although Ca(2+) binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca(2+) binding to the C-terminal lobe of CaM.

Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease

Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.

Linked production of pyroglutamate-modified proteins via self-cleavage of fusion tags with TEV protease and autonomous N-terminal cyclization with glutaminyl cyclase in vivo.

Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities.

Variability in mutational fitness effects prevents full lethal transitions in large quasispecies populations

The distribution of mutational fitness effects (DMFE) is crucial to the evolutionary fate of quasispecies. In this article we analyze the effect of the DMFE on the dynamics of a large quasispecies by means of a phenotypic version of the classic Eigen's model that incorporates beneficial, neutral, deleterious, and lethal mutations. By parameterizing the model with available experimental data on the DMFE of Vesicular stomatitis virus (VSV) and Tobacco etch virus (TEV), we found that increasing mutation does not totally push the entire viral quasispecies towards deleterious or lethal regions of the phenotypic sequence space. The probability of finding regions in the parameter space of the general model that results in a quasispecies only composed by lethal phenotypes is extremely small at equilibrium and in transient times. The implications of our findings can be extended to other scenarios, such as lethal mutagenesis or genomically unstable cancer, where increased mutagenesis has been suggested as a potential therapy.

An evolutionary arms race between plants and viruses (568.2)

Common pokeweed plant (Phytolacca Americana) produces pokeweed antiviral protein (PAP) as a defense mechanism against foreign pathogenic invaders. PAP is a ribosome inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein, and is a potent antiviral agent against many plant, animal, and human viruses. This study aims to elucidate the mechanism of RNA depurination and to understand how PAP recognizes and targets various RNAs. Here, we investigating interactions between PAP and Turnip mosaic virus genome linked protein (VPg). VPg functions as a cap analog in cap-independent translation, and potentially target PAP to uncapped IRES-containing RNA. Fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nM); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate, and competes with structured RNA derived from tobacco etch virus (TEV) for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms, and also suggests the possible use of this protein against the cytotoxic activity of RIPs.

Elongation factor 1A binds the 3’ translation element of the barley yellow dwarf virus (566.4)

The Barley Yellow Dwarf Virus (BYDV) is especially devastating due to its global widespread distribution and its infection of staple cereal crops. Our research aims to understand mechanisms of BYDV mRNA translation to gain insight into combating the disease and its dire economic implications. Previous studies have shown that BYDV initiates translation in a way that is unique to most eukaryotic messages. Almost all eukaryotic mRNA consist of a 5’ cap and initiates translation at the 5’ untranslated region (UTR). BYDV mRNA, however, consists of a cap‐independent translation element at the 3’ UTR (BTE). More interestingly, eukaryotic initiation factor 4F (eI4F) binds the 3’ end of BYDB mRNA, at the BTE, and translation occurs when the 3’ UTR interacts with the 5’ UTR. Pull‐down experiments using the BTE RNA as bait have revealed not only eIF4F binding but also a small array of other proteins not present when a nonfunctional BTE (BTEBF) or the 5’ UTR is used as bait. One prominent protein that appears to bind BTE but not the control BTEBF or 5’ UTR RNA is an unknown 50 kDa protein. Using Mass Spectrometry, we identify this protein to be elongation factor 1A (EF1A). EF1A has previously been shown to bind the 3’ UTR of Tobacco Etch Virus (TEV) mRNA and may have a role in in regulation and replication of the viral genes.Grant Funding Source: NSF MCB 1157632

Characterization of Recombinant ELMOD (Cell Engulfment and Motility Domain) Proteins as GTPase-activating Proteins (GAPs) for ARF Family GTPases

The ARF family of regulatory GTPases, within the RAS superfamily, is composed of ~30 members in mammals, including up to six ARF and at least 18 ARF-like (ARL) proteins. They exhibit significant structural and biochemical conservation and regulate a variety of essential cellular processes, including membrane traffic, cell division, and energy metabolism; each with links to human diseases. We previously identified members of the ELMOD family as GTPase-activating proteins (GAPs) for ARL2 that displayed crossover activity for ARFs as well. To further characterize the GAP activities of the three human ELMODs as GAPs we developed new preparations of each after overexpression in human embryonic kidney (HEK293T) cells. This allowed much higher specific activities and enhanced stability and solubility of the purified proteins. The specificities of ELMOD1-3 as GAPs for six different members of the ARF family were determined and found to display wide variations, which we believe will reveal differences in cellular functions of family members. The non-opioid sigma-1 receptor (S1R) was identified as a novel effector of GAP activity of ELMOD1-3 proteins as its direct binding to either ELMOD1 or ELMOD2 resulted in loss of GAP activity. These findings are critical to understand the roles of ELMOD proteins in cell signaling in general and in the inner ear specifically, and open the door to exploration of the regulation of their GAP activities via agonists or antagonists of the S1R.

A new fusion protein platform for quantitatively measuring activity of multiple proteases

BackgroundRecombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases.ResultsWe tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract.ConclusionThe results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with sufficient substrate specificity.

Specific requirement for translation initiation factor 4E or its isoform drives plant host susceptibility to Tobacco etch virus

BackgroundIn plants, eIF4E translation initiation factors and their eIFiso4E isoforms are essential susceptibility factors for many RNA viruses, including potyviruses. Mutations altering these factors are a major source of resistance to the viruses. The eIF4E allelic series is associated with specific resistance spectra in crops such as Capsicum annum. Genetic evidence shows that potyviruses have a specific requirement for a given 4E isoform that depends on the host plant. For example, Tobacco etch virus (TEV) uses eIF4E1 to infect Capsicum annuum but uses eIFiso4E to infect Arabidopsis thaliana. Here, we investigated how TEV exploits different translation initiation factor isoforms to infect these two plant species.ResultsA complementation system was set up in Arabidopsis to test the restoration of systemic infection by TEV. Using this system, Arabidopsis susceptibility to TEV was complemented with a susceptible pepper eIF4E1 allele but not with a resistant allele. Therefore, in Arabidopsis, TEV can use the pepper eIF4E1 instead of the endogenous eIFiso4E isoform so is able to switch between translation initiation factor 4E isoform to infect the same host. Moreover, we show that overexpressing the pepper eIF4E1 alleles is sufficient to make Arabidopsis susceptible to an otherwise incompatible TEV strain. Lastly, we show that the resistant eIF4E1 allele is similarly overcome by a resistance-breaking TEV strain as in pepper, confirming that this Arabidopsis TEV-susceptibility complementation system is allele-specific.ConclusionWe report here a complementation system in Arabidopsis that makes it possible to assess the role of pepper pvr2-eIF4E alleles in susceptibility to TEV. Heterologous complementation experiments showed that the idiosyncratic properties of the 4E and iso4E proteins create a major checkpoint for viral infection of different hosts. This system could be used to screen natural or induced eIF4E alleles to find and study alleles of interest for plant breeding.

Site‐Selective Protein Immobilization through 2‐Cyanobenzothiazole‐Cysteine Condensation

We described a rapid site-selective protein immobilization strategy on glass slides and magnetic nanoparticles, at either the N or C terminus, by a 2-cyanobenzothiazole (CBT)-cysteine (Cys) condensation reaction. A terminal cysteine was generated at either terminus of a target protein by a combination of expressed protein ligation (EPL) and tobacco etch virus protease (TEVp) digestion, and was reacted with the CBT-solid support to immobilize the protein. According to microarray analysis, we found that glutathione S-transferase immobilized at the N terminus allowed higher substrate binding than for immobilization at the C terminus, whereas there were no differences in the activities of N- and C-terminally immobilized maltose-binding proteins. Moreover, immobilization of TEVp at the N terminus preserved higher activity than immobilization at the C terminus. The success of utilizing CBT-Cys condensation and the ease of constructing a terminal cysteine using EPL and TEVp digestion demonstrate that this method is feasible for site-selective protein immobilization on glass slides and nanoparticles. The orientation of a protein is crucial for its activity after immobilization, and this strategy provides a simple means to evaluate the preferred protein immobilization orientation on solid supports in the absence of clear structural information.

Histone H3 Lysine 14 (H3K14) Acetylation Facilitates DNA Repair in a Positioned Nucleosome by Stabilizing the Binding of the Chromatin Remodeler RSC (Remodels Structure of Chromatin)

Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage.