P1' specificity of the S219V/R203G mutant tobacco etch virus protease.

Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1' autoproteolytic cleavage-resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self-inactivation, but also exhibited greater stability and catalytic efficiency than the wild-type enzyme. An R203G substitution has been reported to further relax the P1' specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1' specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1' amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1' variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme-substrate interactions were analyzed in silico. The results indicate highly similar P1' preferences for both enzymes, many side-chains can be accommodated by the S1' binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.

Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins.

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein-protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein-protein interactions.

Development of a Platform for Noncovalent Coupling of Full Antigens to Tobacco Etch Virus-Like Particles by Means of Coiled-Coil Oligomerization Motifs.

Virus-like particles are excellent inducers of the adaptive immune response of humans and are presently being used as scaffolds for the presentation of foreign peptides and antigens derived from infectious microorganisms for subunit vaccine development. The most common approaches for peptide and antigen presentation are translational fusions and chemical coupling, but some alternatives that seek to simplify the coupling process have been reported recently. In this work, an alternative platform for coupling full antigens to virus-like particles is presented. Heterodimerization motifs inserted in both Tobacco etch virus coat protein and green fluorescent protein directed the coupling process by simple mixing, and the obtained complexes were easily taken up by a macrophage cell line.

Spectroscopic Investigation of the Kinetic Mechanism Involved in the Association of Potyviral VPg with the Host Plant Translation Initiation Factor eIF4E.

The infectious cycle of potyviruses requires the formation of a complex between the viral genome-linked protein VPg and the host eukaryotic translation initiation factor 4E, eIF4E. Mutations associated with plant resistance to potyviruses were previously mapped at the eIF4E surface, while on the virus side, mutations leading to plant resistance breaking were identified within the VPg. In the present study, fluorescence spectroscopy was used to probe the contribution of the VPg intrinsically disordered region bearing amino acids determinant of the resistance breaking, to the VPg-eIF4E binding mechanism. Synthetic peptides encompassing the VPg88-120 central region were found to tightly bind to eIF4E. Fluorescence energy transfer experiments show that, upon binding to eIF4E, the N and C termini of the VPg88-111 fragment move closer to one another, at a distance compatible with a α-helix folding. When the VPg112-120 region, which contains amino acids associated with resistance breakdown, is appended to VPg88-111, the complex formation with eIF4E switches from a single-step to a two-step kinetic model. This study revisits a recent investigation of the VPg-eIF4E complex by specifying the contribution of the VPg central helix and its appended disordered region to VPg association with eIF4E.

Hydrodynamic Behavior of the Intrinsically Disordered Potyvirus Protein VPg, of the Translation Initiation Factor eIF4E and of their Binary Complex.

Protein intrinsic disorder is involved in many biological processes and good experimental models are valuable to investigate its functions. The potyvirus genome-linked protein, VPg, displays many features of an intrinsically disordered protein. The virus cycle requires the formation of a complex between VPg and eIF4E, one of the host translation initiation factors. An in-depth characterization of the hydrodynamic properties of VPg, eIF4E, and of their binary complex VPg-eIF4E was carried out. Two complementary experimental approaches, size-exclusion chromatography and fluorescence anisotropy, which is more resolving and revealed especially suitable when protein concentration is the limiting factor, allowed to estimate monomers compaction upon complex formation. VPg possesses a high degree of hydration which is in agreement with its classification as a partially folded protein in between a molten and pre-molten globule. The natively disordered first 46 amino acids of eIF4E contribute to modulate the protein hydrodynamic properties. The addition of an N-ter His tag decreased the conformational entropy of this intrinsically disordered region. A comparative study between the two tagged and untagged proteins revealed the His tag contribution to proteins hydrodynamic behavior.

Enhanced production of soluble tumor necrosis factor-related apoptosis-inducing ligand in Escherichia coli using a novel self-cleavable tag system Fh8-ΔI-CM.

Escherichia coli is an essential host for large-scale expression of heterologous polypeptides. However, further applications are limited by the formation of potential protein aggregates. In this work, we developed a novel on-column tag removal and purification system based on Fh8 hydrophobic interaction chromatography purification and ΔI-CM self-cleavage to obtain soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We evaluated several methods to improve TRAIL solubility and finally demonstrated that the Fh8 tag was a powerful solubility enhancer. Finally, we replaced the tobacco etch virus (TEV) protease site with a ΔI-CM self-cleavage intein to simplify the purification process. The released soluble TRAIL purity and yield reached 98.4% and 82.1 mg/L in shake flasks, respectively. Thus, the Fh8-ΔI-CM system enhanced target protein solubility by Fh8, enabled on-column tag removal and purification based on Fh8 calcium-binding properties and ΔI-CM self-cleavage properties, and promoted the release of highly active protein with high yield and purity. Overall, our findings suggest that this Fh8-ΔI-CM system could be used as a novel solubility-inducing and purification fusion tag for protein production in E. coli.

[Structure of Potato Virus A Coat Protein Particles and Their Dissociation].

This paper reports on a complex structural analysis of the potato virus A coat protein using a set of complementary physico-chemical methods. We have demonstrated previously that this protein does not exist as individual subunits in solution and undergoes association into oligomers with subsequent transition to ß-conformation. The purpose of the present work was to study the possible mechanisms of this transformation and to search for methods that dissociate protein oligomers. To analyze the low resolution protein structure in solution, small-angle X-ray scattering was used. Stable particles representing clusters of 30 coat protein subunits were present even in an aqueous salt solution with a high ionic strength and pH (pH 10.5; 0.5 M NaCl). The particles did not dissociate in the presence of 10 mM dextran sulfates (15 and 100 kDa). Dissociation in the presence of 5.2 mM sodium dodecyl sulfate results in the formation of the subunit-detergent complexes consisting of 10-12 small particles joined together like "beads on a string". Similar effects of sodium dodecyl sulfate were shown for serum albumins (bovine and human). Denaturation of the potato virus A coat protein molecules occurs in the presence of detergent concentrations that are seven times lower than that in albumins (5.2 and 35 mM), which confirms low stability of the potato virus A coat protein. Using spectral methods, preservation of the secondary structure and loss of the tertiary structure of the protein in its complex with sodium dodecyl sulfate have been demonstrated. Possible mechanism for protein particle formation through the interaction between unordered terminal domains and their transformation into ß-structures has been suggested.

Biological and molecular characterization of a highly divergent johnsongrass mosaic virus isolate from Pennisetum purpureum.

The complete genome sequence (9,865 nucleotides) of a highly divergent johnsongrass mosaic virus isolate (JGMV-CNPGL) was determined using Illumina sequencing. This isolate infected 10 genotypes of gramineous plants including maize. A comparative analysis of the complete genome showed 80 % nucleotide (nt) sequence identity (86 % amino acid (aa) sequence identity) to a johnsongrass mosaic virus isolate from Australia. The coat protein (CP) identity values, however, were lower than those for the whole genome (78 % and 80 % for nt and aa, respectively) and were close to the species demarcation values (77 % nt and 80 % aa). Unexpectedly, the amino-terminal portion of CP of JGMV-CNPGL showed only 38 % sequence identity to other JGMV isolates. The biological implications of this sequence divergence remain to be elucidated.

Surface plasmon resonance for monitoring the interaction of Potato virus Y with monoclonal antibodies.

Surface plasmon resonance (SPR)-based biosensors have been widely utilized for measuring interactions of a variety of molecules. Fewer examples include higher biological entities such as bacteria and viruses, and even fewer deal with plant viruses. Here, we describe the optimization of an SPR sensor chip for evaluation of the interaction of the economically relevant filamentous Potato virus Y (PVY) with monoclonal antibodies. Different virus isolates were efficiently and stably bound to a previously immobilized polyclonal antibody surface, which remained stable over subsequent injection regeneration steps. The ability of the biosensor to detect and quantify PVY particles was compared with ELISA and RT-qPCR. Stably captured virus surfaces were successfully used to explore kinetic parameters of the interaction of a panel of monoclonal antibodies with two PVY isolates representing the main viral serotypes N and O. In addition, the optimized biosensor proved to be suitable for evaluating whether two given monoclonal antibodies compete for the same epitope within the viral particle surface. The strategy proposed in this work can help to improve existing serologic diagnostic tools that target PVY and will allow investigation of the inherent serological variability of the virus and exploration for new interactions of PVY particles with other proteins.

Complete genome sequence of Habenaria mosaic virus, a new potyvirus infecting a terrestrial orchid (Habenaria radiata) in Japan.

The complete genomic sequence of Habenaria mosaic virus (HaMV), which infects terrestrial orchids (Habenaria radiata), has been determined. The genome is composed of 9,499 nucleotides excluding the 3'-terminal poly(A) tail, encoding a large polyprotein of 3,054 amino acids with the genomic features typical of a potyvirus. Putative proteolytic cleavage sites were identified by sequence comparison to those of known potyviruses. The HaMV polyprotein showed 58 % amino acid sequence identity to that encoded by the most closely related potyvirus, tobacco vein banding mosaic virus. Phylogenetic analysis of the polyprotein amino acid sequence and its coding sequences confirmed that HaMV formed a cluster with the chilli veinal mottle virus group, most of which infect solanaceous plants. These results suggest that HaMV is a distinct member of the genus Potyvirus.

Analysis of the complete sequences of two biologically distinct Zucchini yellow mosaic virus isolates further evidences the involvement of a single amino acid in the virus pathogenicity.

The complete genome sequences of two Slovak Zucchini yellow mosaic virus isolates (ZYMV-H and ZYMV-SE04T) were determined. These isolates differ significantly in their pathogenicity, producing either severe or very mild symptoms on susceptible cucurbit hosts. The viral genome of both isolates consisted of 9593 nucleotides in size, and contained an open reading frame encoding a single polyprotein of 3080 amino acids. Despite their different biological properties, an extremely high nucleotide identity could be noted (99.8%), resulting in differences of only 5 aa, located in the HC-Pro, P3, and NIb, respectively. In silico analysis including 5 additional fully-sequenced and phylogenetically closely-related isolates known to induce different symptoms in cucurbits was performed. This suggested that the key single mutation responsible for virus pathogenicity is likely located in the N-terminal part of P3, adjacent to the PIPO.

Resistance of non-transgenic papaya plants to papaya ringspot virus (PRSV) mediated by intron-containing hairpin dsRNAs expressed in bacteria.

RNA-mediated virus resistance based on natural antiviral RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants. In this study, a conserved 3'-region (positions 9839-10117, 279 nt) of the capsid protein (CP) gene of papaya ringspot virus (PRSV), designated CP279, was used to generate an intron-containing hairpin RNA (ihpRNA) construct by one-step, zero-background ligation-independent cloning (OZ-LIC). The RNaseIII-deficient Escherichia coli strain M-JM109lacY was identified as the best choice for producing large quantities of specific ihpRNA-CP279. Resistance analyses and ELISA data verified that most papaya plants mechanically co-inoculated with TRIzol-extracted ihpRNA-CP279 and PRSV were resistant to PRSV, and resistance was maintained throughout the test period (>2 months post-inoculation). In contrast, a 1-2 day interval between sequential inoculation of PRSV and ihpRNA-CP279 did not result in complete protection against PRSV infection, but delayed the appearance of viral symptoms by 3 to 4 days. These findings indicate that direct mechanical inoculation of papaya plants with bacterially-expressed ihpRNA-CP279 targeting the PRSV CP gene can interfere with virus infection. This work lays a foundation for developing a non-transgenic approach to control PRSV by directly spraying plants with ihpRNA or crude bacterial extract preparations.

Sequence variation of P1 gene in Potato virus Y isolated from Fujian Province.

To understand the sequence variation and the putative protein structure of P1 gene in Potato virus Y (PVY) and to identify the sources of the variation, P1 gene in PVY isolated from Fujian Province was amplified by reverse-transcription polymerase chain reaction (RT-PCR) using a pair of degenerate primers designed from the conserved regions of published sequences. Sequence variation and putative protein structure were analyzed, and phylogenetic tree was reconstructed using Bayesian inference method. Expected fragments of 915 bp in size were amplified from 12 samples collected from Fujian Province by RT-PCR. The 12 sequences shared 73%-99% nucleotide identity with the reference sequences from GenBank. A strong recombination signal was identified at position 309 in sequences of isolates QK44, XT02, XT08 and LH05. Among the 12 sequences, 85 amino acid variants were detected, indicating high sequence variation in the P1 protein. However, positions 41-275 in the protein were highly conserved, especially in three active sites (H192, D201 and V235). Phylogenetic analysis grouped the sequences into three clades, each with different Coiled-coil domains and 3D-structures, suggesting divergent phylogenetic relationship among the groups. The above results show P1 gene in PVY is highly variable but contains 3 conserved active sites (H192, D201, V235) and the high genetic variation in the gene is primarily due to mutation and recombination.

Discovery of Pyrido[1,2-α] Pyrimidinone Mesoionic Compounds as Potential Control Agents Against Potato Virus Y.

Potato virus Y (PVY) relies on aphids and tubers to spread in the field and causes serious economic losses in the potato industry. Here, we found that pyrido[1,2-α] pyrimidinone mesoionic compounds with insecticidal activity against aphids possessed a good inhibitory effect on PVY. Among them, compound 35 had the best inhibitory activity against PVY (EC50 = 104 µg/mL), even superior to that of ningnanmycin (125 µg/mL). The fluorescence and qPCR results confirmed that compound 35 could inhibit the proliferation of PVY in Nicotiana benthamiana. Preliminary experiments on the mechanism of action indicated that compound 35 had good binding affinity with the coat protein (CP), which plays an essential role in aphid-PVY interactions. Molecular docking revealed that compound 35 could bind to the pocket of CP formed by Ser52, Glu204, and Arg208. Compound 35 had substantially lower binding affinity (Kd) values with CPS52A (219 µM), CPE204A (231 µM), and CPR208A (189 µM) than those with CPWT (5.80 µM). A luciferase assay confirmed that mutating Ser52, Glu204, and Arg208 significantly affected the expression level of CP and further reduced virus proliferation. Therefore, the broad-spectrum activity of compound 35 provides a unique strategy for the prevention and treatment of PVY.

The C terminus of lettuce mosaic potyvirus cylindrical inclusion helicase interacts with the viral VPg and with lettuce translation eukaryotic initiation factor 4E.

Recessive resistance to lettuce mosaic virus (LMV) is conferred in lettuce by the mo1 gene, encoding the eukaryotic translation initiation factor 4E (eIF4E). The C terminus of the viral cylindrical inclusion helicase (CI-Cter), together with the VPg, is involved directly in overcoming mo1 resistance. In this study, recombinant LMV VPg and CI-Cter proteins from wild-type or resistance-breaking isolates were expressed and purified from Escherichia coli. The allelic forms of eIF4E from susceptible or resistant lettuce cultivars were produced similarly and these proteins were used in ELISA-based assays to demonstrate the in vitro binding of the various forms of LMV CI-Cter to both lettuce eIF4E and LMV VPg proteins. All combinations tested displayed significant and specific interactions, and the interaction between the C-terminal part of the LMV CI and eIF4E was confirmed in vivo in bimolecular fluorescence complementation assays. Higher interaction signals for both CI-eIF4E and CI-VPg were observed for LMV-E, indicating that the eIF4E interaction network involving CI and VPg appears to be stronger in the case of this resistance-breaking isolate. This could suggest the need for a minimal interaction threshold for infection success in resistant lettuce, but more precise measurement of the interaction parameters linking eIF4E, VPg and CI is needed in order to reinforce such a hypothesis.

Structural flexibility allows the functional diversity of potyvirus genome-linked protein VPg.

Several viral genome-linked proteins (VPgs) of plant viruses are intrinsically disordered and undergo folding transitions in the presence of partners. This property has been postulated to be one of the factors that enable the functional diversity of the protein. We created a homology model of Potato virus A VPg and positioned the known functions and structural properties of potyviral VPgs on the novel structural model. The model suggests an elongated structure with a hydrophobic core composed of antiparallel ß-sheets surrounded by helices and a positively charged contact surface where most of the known activities are localized. The model most probably represents the fold induced immediately after binding of VPg to a negatively charged lipid surface or to SDS. When the charge of the positive surface was lowered by lysine mutations, the efficiencies of in vitro NTP binding, uridylylation reaction, and unspecific RNA binding were reduced and in vivo the infectivity was debilitated. The most likely uridylylation site, Tyr63, locates to the positively charged surface. Surprisingly, a Tyr63Ala mutation did not prevent replication completely but blocked spreading of the virus. Based on the localization of Tyr119 in the model, it was hypothesized to serve as an alternative uridylylation site. Evidence to support the role of Tyr119 in replication was obtained which gives a positive example of the prediction power of the model. Taken together, our experimental data support the features presented in the model and the idea that the functional diversity is attributable to structural flexibility.

Immune response to a potyvirus with exposed amino groups available for chemical conjugation.

BACKGROUND: The amino terminus of the tobacco etch virus (TEV) capsid protein is located on the external surface of infectious TEV particles, as proposed by previous studies and an in silico model. The epsilon amino groups on the exposed lysine residues are available for chemical conjugation to any given protein, and can thus act as antigen carriers. The availability of amino groups on the surfaces of TEV particles was determined and the immune response to TEV evaluated. RESULTS: Using a biotin-tagged molecule that reacts specifically with amino groups, we found that the TEV capsid protein has amino groups on its surface available for coupling to other molecules via crosslinkers. Intraperitoneal TEV was administered to female BALB/c mice, and both their humoral and cellular responses measured. Different IgG isotypes, particularly IgG2a, directed against TEV were induced. In a cell proliferation assay, only spleen cells from vaccinated mice that were stimulated in vitro with TEV showed significant proliferation of CD3+/CD4+ and CD3+/CD8+ subpopulations and secreted significant amounts of interferon γ. CONCLUSIONS: TEV has surface amino groups that are available for chemical coupling. TEV induces both humoral and cellular responses when administered alone intraperitoneally to mice. Therefore, TEV should be evaluated as a vaccine adjuvant when chemically coupled to antigens of choice.

Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus.

The multifunctional helper component proteinase (HC-Pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (P1) and NIa proteinase, processes the polyprotein into mature proteins. In this study, we analysed the autoproteolytic active domain of zucchini yellow mosaic virus (ZYMV) HC-Pro. Several Escherichia coli-expressed MBP:HC-Pro:GFP mutants containing deletions or point mutations at either the N- or C-terminus of the HC-Pro protein were examined. Our results showed that amino acids essential for the proteolytic activity of ZYMV HC-Pro are distinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses.

Is the 6 kDa tobacco etch viral protein a bona fide ERES marker?

The claim that the 6 kDa viral protein (VP) of Tobacco Etch Virus is a marker for ER exit sites (ERES) has been investigated. When transiently expressed as a CFP tagged fusion construct in tobacco mesophyll protoplasts, this integral membrane protein co-localizes with both the COPII coat protein YFP-SEC24 and the Golgi marker Man1-RFP. However, when over-expressed the VP locates to larger spherical structures which co-localize with neither ER nor Golgi markers. Nevertheless, deletion of the COPII interactive N-terminal D(X)E motif causes it to be broadly distributed throughout the ER, supporting the notion that this protein could be an ERES marker. Curiously, whereas brefeldin A (BFA) caused a typical Golgi-stack response (redistribution into the ER) of the VP in leaf epidermal cells, in protoplasts it resulted in the formation of structures identical to those formed by over-expression. However, anomalous results were obtained with protoplasts: when co-expressed with the non-cycling cis-Golgi marker Man1-RFP, a BFA-induced redistribution of the VP-CFP signal into the ER was observed, but, in the presence of the cycling Golgi marker ERD2-YFP, this did not occur. High resolution images of side-on views of Golgi stacks in epidermal cells showed that the 6 kDa VP-CFP signal overlapped considerably more with YFP-SEC24 than with Man1-RFP, indicating that the VP is proportionately more associated with ERES. However, based on a consideration of the structure of its cytoplasmic tail, the scenario that the VP collects at ERES and is transported to the cis-Golgi before being recycled back to the ER, is supported.

Indigenous and introduced potyviruses of legumes and Passiflora spp. from Australia: biological properties and comparison of coat protein nucleotide sequences.

Five Australian potyviruses, passion fruit woodiness virus (PWV), passiflora mosaic virus (PaMV), passiflora virus Y, clitoria chlorosis virus (ClCV) and hardenbergia mosaic virus (HarMV), and two introduced potyviruses, bean common mosaic virus (BCMV) and cowpea aphid-borne mosaic virus (CAbMV), were detected in nine wild or cultivated Passiflora and legume species growing in tropical, subtropical or Mediterranean climatic regions of Western Australia. When ClCV (1), PaMV (1), PaVY (8) and PWV (5) isolates were inoculated to 15 plant species, PWV and two PaVY P. foetida isolates infected P. edulis and P. caerulea readily but legumes only occasionally. Another PaVY P. foetida isolate resembled five PaVY legume isolates in infecting legumes readily but not infecting P. edulis. PaMV resembled PaVY legume isolates in legumes but also infected P. edulis. ClCV did not infect P. edulis or P. caerulea and behaved differently from PaVY legume isolates and PaMV when inoculated to two legume species. When complete coat protein (CP) nucleotide (nt) sequences of 33 new isolates were compared with 41 others, PWV (8), HarMV (4), PaMV (1) and ClCV (1) were within a large group of Australian isolates, while PaVY (14), CAbMV (1) and BCMV (3) isolates were in three other groups. Variation among PWV and PaVY isolates was sufficient for division into four clades each (I-IV). A variable block of 56 amino acid residues at the N-terminal region of the CPs of PaMV and ClCV distinguished them from PWV. Comparison of PWV, PaMV and ClCV CP sequences showed that nt identities were both above and below the 76-77% potyvirus species threshold level. This research gives insights into invasion of new hosts by potyviruses at the natural vegetation and cultivated area interface, and illustrates the potential of indigenous viruses to emerge to infect introduced plants.