- Open Access
Intrinsic disorder in Viral Proteins Genome-Linked: experimental and predictive analyses
© Hébrard et al; licensee BioMed Central Ltd. 2009
Received: 26 January 2009
Accepted: 16 February 2009
Published: 16 February 2009
VPgs are viral proteins linked to the 5' end of some viral genomes. Interactions between several VPgs and eukaryotic translation initiation factors eIF4Es are critical for plant infection. However, VPgs are not restricted to phytoviruses, being also involved in genome replication and protein translation of several animal viruses. To date, structural data are still limited to small picornaviral VPgs. Recently three phytoviral VPgs were shown to be natively unfolded proteins.
In this paper, we report the bacterial expression, purification and biochemical characterization of two phytoviral VPgs, namely the VPgs of Rice yellow mottle virus (RYMV, genus Sobemovirus) and Lettuce mosaic virus (LMV, genus Potyvirus). Using far-UV circular dichroism and size exclusion chromatography, we show that RYMV and LMV VPgs are predominantly or partly unstructured in solution, respectively. Using several disorder predictors, we show that both proteins are predicted to possess disordered regions. We next extend theses results to 14 VPgs representative of the viral diversity. Disordered regions were predicted in all VPg sequences whatever the genus and the family.
Based on these results, we propose that intrinsic disorder is a common feature of VPgs. The functional role of intrinsic disorder is discussed in light of the biological roles of VPgs.
The interactions between eukaryotic translation initiation factors eIF4Es and Viral proteins genome-linked (VPgs) are critical for plant infection by potyviruses (for review see ). Mutations in plant eIF4Es result in recessive resistances [2–7]. Mutations in VPgs of several potyviruses result in resistance-breaking isolates [7–14]. These interactions were demonstrated in vitro by interaction assays and in planta by mean of co-localisation experiments [15–22]. Their exact roles are still unclear, although VPg/eIF4E interactions had been suggested to be involved in protein translation, in RNA replication and in cell-to-cell movement (for review see ). A similar interaction has been postulated in the rice/Rice yellow mottle virus (RYMV, Sobemovirus) pathosystem, involving the virulence factor VPg and the resistance factor eIF(iso)4G .
Recently, Sesbania mosaic virus (SeMV, genus Sobemovirus), Potato virus Y (PVY, genus Potyvirus) and Potato virus A (PVA, genus Potyvirus) VPgs were reported to be "natively unfolded proteins" [25–27]. Natively unfolded proteins, also called intrinsically disordered proteins (IDPs), lack a unique 3D-structure and exist as a dynamic ensemble of conformations at physiological conditions. Proteins may be partially or fully intrinsically disordered, possessing a wide range of conformations depending on the degree of disorder. Disordered domains have been grouped into at least two broad classes – compact (molten globule-like) and extended (natively unfolded proteins) [28, 29]. IDPs possess a number of crucial biological functions including molecular recognition and regulation [30–37]. The functional diversity provided by disordered regions is believed to complement functions of ordered protein regions by protein-protein interactions [38–40].
Intrinsically unstructured proteins and regions differ from structured globular proteins and domains with regard to many attributes, including amino acid composition, sequence complexity, hydrophobicity, charge, flexibility, and type and rate of amino acid substitutions over evolutionary time. Many of these differences were utilized to develop various algorithms for predicting intrinsic order and disorder from amino acid sequences [41, 42]. Bioinformatic analyses using disorder predictors showed that a surprisingly high percentage of genome putative coding sequences are intrinsically disordered. Eukaryotes genomes would encode more disordered proteins than prokaryotes having 52–67% of their translated products containing segments predicted to have more than 40 consecutive disordered residues [43–47]. The highest proportion of conserved predicted disordered regions (PDRs) is found in protein domains involved in protein-protein transient interactions (signalling and regulation). So far, disorder prediction data for viral proteins are scarce, although viruses have been shown to contain the highest proportion of proteins containing conserved predicted disordered regions (PDRs) compared to archaea, bacteria and eukaryota .
The presence of VPgs is not restricted to poty- and sobemoviruses but is also found in animal viruses with double or positive single strand (ss) RNA genome belonging to several unrelated virus families and genera. The term "VPg" refers to proteins highly diverse in sequence and in size (2–4 kDa for Picornaviridae and Comoviridae members, 10–26 kDa for Potyviridae, Sobemoviruses and Caliciviridae members, and up to 90 kDa for Birnaviridae members) . High-resolution structural data are limited to 2–4 kDa VPgs. The 3D structures of synthetic peptides corresponding to Picornaviridae VPgs are the only ones available to date [49–51].
In this paper, we report the bacterial expression, purification and biochemical characterization of VPgs from Rice yellow mottle virus (RYMV) and Lettuce mosaic virus (LMV), two viruses of agronomic interest related to SeMV (genus Sobemovirus) or PVY and PVA (genus Potyvirus). We show that they both contain disordered regions although at a different extent. We next extend these results to a set of 14 VPg sequences representative of the various viral species. In particular, we focused on viruses for which functional VPg domains have been mapped, and in particular to those viruses the VPgs of which are known to interact with translation initiation factors. The disorder propensities of the 14 VPg sequences were assessed in silico using several complementary disorder predictors. Finally, the possible implications of structural disorder of VPgs in light of to their biological functions are discussed.
Experimental evidences of intrinsic disorder in RYMV and LMV VPgs
Previous secondary structure predictions have suggested that both RYMV and LMV VPgs contain a high proportion of α-helices, 35% and 33% respectively [21, 24]. The secondary structure stabilizer 2,2,2-trifluoroethanol (TFE) was therefore used to test the propensity of these proteins to undergo induced folding into an α-helical conformation. The gain of α-helicity by both VPgs, as judged based on the characteristic maximum at 190 nm and minima at 208 and 222 nm, parallels the increase in TFE concentration (Figure 2). The α-helical propensity of VPgs is revealed at TFE concentrations as low as 5%. Further calculations carried out with the K2d program  indicated an α-helix content of 30% (± 4%) for RYMV VPg in the presence of 30% TFE.
Disorder predictions in sobemoviral VPgs
Disorder predictions in potyviral VPgs
Disorder predictions in caliciviral VPgs
Often, intrinsically disordered regions involved in protein-protein interactions and molecular recognition undergo disorder-to-order transitions upon binding [30–32, 35, 59–63]. A correlation has been established between the specific pattern in the PONDR® VLXT curve and the ability of a given short disordered regions to undergo disorder-to-order transitions on binding . Based on these specific features, an α-MoRF predictor was recently developed [60, 65].
Location of predicted α-MoRFs in VPgs
CDF and CH-plot analyses
In this paper, we provide experimental evidences that RYMV and LMV VPgs contain intrinsically disordered regions. These findings, together with the previous reports documenting the disordered state of SeMV, PVY and PVA VPgs [25–27], suggest that intrinsic disorder may be a common and distinctive feature of sobemo- and potyviral VPgs. By carrying out an in-depth in silico analysis, we show that the disordered state of VPgs depend on the viral genera. Sobemoviral SeMV and RYMV VPgs appeared highly disordered with (i) 30% and 50% increases of their molecular masses estimated from SDS-PAGE compared to expected masses, respectively, and (ii) far-UV CD spectra with large negative ellipticities near 200 nm and low ellipticities at 190 nm. By contrast, the increase of the apparent molecular masses of potyviral VPgs from SDS-PAGE are moderate (<5% for LMV, approx. 10% for PVY and PVA) and the trends of far-UV CD spectra indicate partial disorder better suggesting short disordered regions included in globally ordered VPgs.
The experimentally observed disorder is also pointed out by complementary in silico analyses. However, quantitative assessment of disorder prediction strengths and precise location of consensus disordered regions turned out to be hectic. While LMV, PVY and PVA VPgs showed longer disordered segments, SeMV VPg showed short disordered segments whereas experimental results were similar to RYMV VPg. Moreover, binary predictors which are intended to allow a comparison of relative disordered states failed to detect disorder in several VPgs, including those for which the disordered state has been shown experimentally such as SeMV and PVA. However, it is important to notice that these predictors are meant to predict disorder on an entire protein basis, and SeMV and PVA not only have substantial ordered regions, but their disordered regions are in general shorter than those of the other proteins studied. These features could have easily tipped the balance towards an "ordered protein" prediction. Otherwise, the use of complementary disorder predictors induces difficulties to precisely map consensus disordered regions in VPgs, but this is due mainly to the fact that different disorder predictors are built upon slightly different definitions of disorder . This is what makes these predictions complementary of each other.
The presence of intrinsically disordered (ID) regions was detected by five per-residue disorder predictors in 10–26 kDa VPgs. At intra-specific level in sobemo- and in potyviruses, the presence of intrinsic disorder regions was conserved independently from sequence conservation. Therefore, we enlarged our analysis to other genera, namely caliciviral VPgs that had never been suggested before to be disordered, and small VPgs (2 to 3 kDa) from Picornaviridae and Comoviridae where ID was also predicted (data not shown). By contrast to several domains in capsid and polymerase viral proteins, the disorder propensity had not been described so far as a common property of VPgs . The methodology used by Chen and colleagues is likely not adapted to the highly diverse set of VPg sequences because it includes a first step of conserved domain identification before performing the disorder predictions.
VPg ID was rather predicted in several small patches (<30 residues) than in few large domains, this trend is common in short protein sequences with binding sites. These characteristics of variable degree of disorder, together with the complementarities of disorder definitions described above, may explain why discrepancies in location of PDRs were frequently observed. Still, all proteins showed a high predicted disorder content (percentage of disordered residues), ranging in average from 44% for sobemoviral to 60% for caliciviral VPgs (PONDR® VSL2 predictions). Part of the hydrophobic residues of VPgs would be involved in the formation of additional secondary structure elements. We performed in silico detection of α-helix-forming molecular recognition features (α-MoRF) which mediate the binding of initially disordered domains with interaction partners . Some α-MoRF domains were detected in the N-terminal regions of VPgs which were not reported to be interacting domains. By contrast, the first half of the C-terminal domain of RYMV VPg and the central domain of LMV VPg previously predicted to form α-helices [21, 24] were not identified as α-MoRFs. These domains were predicted both to be disordered and to form α-helices. The α-helical propensities of RYMV VPgs, as observed in the presence of TFE concentration as low as 5% (Figure 2), suggest that some disordered regions in the isolated proteins may undergo a disorder-to-order transition upon association with a partner protein. Noteworthy, the only VPg structures available to date (Picornaviridae) were obtained either in the presence of a stabilizing agent  or in association with the viral RNA-dependent RNA polymerase (3D) which probably stabilized the VPg folded state [50, 51].
The property of proteins to be intrinsically disordered confers to them the ability to bind to many different partners. These characteristics likely explain why many proteins critical in interaction networks (hub proteins) are intrinsically disordered [36, 45]. In RYMV VPg, the resistance-breaking positions 48 and 52 suggested to be involved in eIF(iso)4G interaction are located in a putative α-helix also predicted to be disordered. The same result is obtained with LMV VPg where resistance-breaking sites involved in eIF4E interaction are located in the central domain predicted to contain two α-helices and to display disorder features. Analysis of other potyviral VPgs suggests that domains associated with virulence are often disordered with some residual structure. Besides their interactions with eIF4Es, potyviral VPgs were found to interact with a variety of host factors such as poly(A)-binding protein [68, 69], eIF4G  and eukaryotic elongation factor eEF1A . Multiple in vitro interactions of VPgs with eIF4GI , eIF3  and eIF4A , and others proteins belonging to the translation initiation complex, were also shown for Caliciviridae members. Potyviral VPgs were also reported to interact with several viral proteins such as NIb, HC-Pro, CI and CP [9, 68, 74].
As underlined in the introduction, VPgs are multifunctional proteins. At least part of their functions implies interactions with eIFs, with the VPg/eIF4E interaction having been shown to enhance the in vitro translation of viral RNA [22, 75]. VPgs were suggested to mimic the mRNA 5'-linked cap recruiting the translation initiation complex. Besides, a ribonuclease activity of VPgs was reported. It might contribute to host RNA translation shutoff . VPg-eIF interactions were also suggested to be involved in other key steps in the viral cycle . In Picornaviridae, it was established that VPg is involved in genome replication, its uridyl-form acting as primer for complementary strand synthesis [77, 78]. An additional role of potyviral VPg-eIF4E interactions in plant cell-to-cell movement via eIF4G and microtubules was also suggested [2, 79]. VPg could participate to a putative vascular movement complex to cross the plasmodesmata and may facilitate virus unloading [9, 80]. Thus, VPg might be involved in key steps of the viral cycle such as replication, translation and movement. Additionally, ID VPg was reported to be necessary to the processing of SeMV polyprotein by viral protease . ID might explain how a unique protein can perform and regulate these different biological functions. PDRs might give to the VPg the necessary plasticity to fit surface overlaps with various partners.
Experimentally, we showed that RYMV and LMV VPgs contain both intrinsically disordered domains but with different disordered states. Using in silico analyses, ID domains were predicted to occur in 14 VPgs of sobemo-, poty-and caliciviruses. Although highly diverse, VPgs share the common feature of possessing ID domains. These structural properties of VPgs are more conserved than what could be anticipated from their sequence homologies. However, comparative analyses at intra-and interspecies levels showed the diversity of intrinsic disorder in VPgs.
Like many IDPs, VPg ID domains may play a role in protein interaction networks, interacting in particular with translation initiation factor eIFs to perform key steps of the viral cycle (replication, translation and movement).
Purification of recombinant RYMV and LMV VPgs
The VPg-encoding region in the RYMV ORF2a was amplified by PCR from FL5 infectious clone  by using the primers FCIaVPgH 5'ATATCCATGGGATCCCA TTTGAGATTTACGGC (containing a Nco I site and RYMV nucleotides 1587–1607) and RCIaVPgH 5'TGCAAGATCTCTCGATATCAACATCCTCGCC (containing a Bgl II site and sequence complementary to RYMV nucleotides 1823–1803). The resulting fragment was cloned into the Nco I and Bgl II sites of pQE60 as a 6-His C-terminal fusion (Qiagen) and the construct was sequenced. The resulting expression plasmid was used to transform the E. coli strain M15-pRep4 (Qiagen). After induction with 0.5 mM isopropyl-1-thio-β-D-galactopyranoside at 25°C for 5 h, the cells from 1 L culture in LB medium were harvested by centrifugation and frozen at -80°C. Cells were thawn, resuspended in 30 mL of purification buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol), disrupted with a French press (Thermo) and centrifuged at 18000 rpm for 30 min. The supernatant was filtered (0.5 μm filters) and purification of the VPg in native conditions was carried out using a nickel-loaded HiTrap IMAC HP column (GE Healthcare) followed by gel filtration step onto a HR10/30 Superdex 75 column (GE Healthcare) in 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5% glycerol.
LMV VPg was produced in E. coli using the pTrcHis plasmid as expression vector as already described . The N-terminal His-tagged protein was found to be expressed in the soluble fraction of the bacterial lysate and was purified as described above, except that 50 mM Tris-HCl pH 8, 800 mM NaCl, 10% glycerol, 2 mM β-mercaptoethanol was used as the affinity chromatography buffer, and 20 mM Tris-HCl pH 8, 800 mM NaCl, 5% glycerol as gel filtration buffer.
Circular dichroism analyses
Freshly purified protein samples were used for CD analyses. Sample buffer was changed by eluting the protein from a PD10 desalting column (GE Healthcare) using 10 mM sodium phosphate buffer (pH 8.0), supplemented with 300 mM or 500 mM NaF for RYMV or LMV VPgs respectively. After centrifugation, the protein concentration was determined using a ND-1000 Spectrophotometer (NanoDrop Technologies) and an extinction coefficient of 7,780 and 18,490 M-1cm-1 for RYMV and LMV VPgs respectively. Far UV-CD spectra were recorded with a chirascan dichrograph (Applied Photophysics) in a thermostated (20°C) quartz circular cell with a 0.5 mm path length, in steps of 0.5 nm. All protein spectra were corrected by subtraction of the respective buffer spectra. The mean molar ellipticity values per residue were calculated using the manufacturer software. Structural variations of the native protein samples were monitored by recording successive CD spectra after addition of 2,2,2-trifluoroethanol (TFE, Sigma) in the 5–30% range (vol:vol).
Sequences for this study were obtained from the viral genome resources at NCBI http://www.ncbi.nlm.nih.gov/genomes/genlist.cgi?taxid=10239type=5name=Viruses. Sequence accession numbers are: Sobemovirus (RYMV AJ608219, CoMV NC_002618, RGMoV NP_736586, SBMV NP_736583, SCPMV NP_736598, SeMV NP_736592), Potyvirus (LMV NP_734159, PVY NP_734252, PVA NC_004039, TEV NP_734204, TuMV NC_002509, BYMV NC_003492), and Caliciviridae (RHDV NP_740330, VESV NP_786894, SV Man X86560, NV NP_786948).
Seven programs were used to predict the disorder tendency of VPgs. PONDR®, Predictors of Natural Disordered Regions, version VLXT is a neural network principally based on local amino acid composition, flexibility and hydropathy http://www.pondr.com. FoldIndex© is based on charge and hydropathy analyzed locally using a sliding window http://bip.weizmann.ac.il/fldbin/findex. DISOPRED2 is also a neural network, but incorporates information from multiple sequence alignments generated by PSI-BLAST http://bioinf.cs.ucl.ac.uk/disopred. PONDR® VSL2 has achieved higher accuracy and improved performance on short disordered regions, while maintaining high performance on long disordered regions http://www.ist.temple.edu/disprot/predictorVSL2.php. IUPred uses a novel algorithm that evaluates the energy resulting from inter-residue interactions http://iupred.enzim.hu. PONDR® VLXT and VSL2 as well as DISOPRED2 were all trained on datasets of disordered proteins, while FoldIndex© and IUPred were not. Binary classifications of VPgs as ordered or disordered were performed using CDF and CH-plot analyses. Cumulative distribution function curves or CDF curves were generated for each dataset using PONDR® VLXT scores for each of the VPgs . Charge-hydropathy distributions (CH-plots) were also analyzed using the method described in Uversky et al. .
The predictor of α-helix forming Molecular Recognition Features, α-MoRF, focuses on short binding regions within regions of disorder that are likely to form helical structure upon binding [60, 65]. It utilizes a stacked architecture, where PONDR® VLXT is used to identify short predictions of order within long predictions of disorder and then a second level predictor determines whether the order prediction is likely to be a binding site based on attributes of both the predicted ordered region and the predicted surrounding disordered region. An α-MoRF prediction indicates the presence of a relatively short (20 residues), loosely structured helical region within a largely disordered sequence [60, 65]. Such regions gain stable structure upon a disorder-to-order transition induced by binding to partner.
We are grateful to Anne-Lise Haenni and Jean-François Laliberté for helpful discussions. We thank Jean-Paul Brizard for technical advice.
This work was partially supported by the French National Agency for Research ('Poty4E', ANR-05-Blan-0302-01).
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