Intrinsic disorder in Viral Proteins Genome-Linked: experimental and predictive analyses

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Electrophoretic mobility and size-exclusion chromatography profile of RYMV and LMV VPgs. A, C. 15% SDS-PAGE of recombinant His-tagged RYMV and LMV VPgs recovered from the supernatant (SN) and from the cell pellet (CP) after E. coli cell extraction, and after imidazole gradient elution fractions (E1 to E5) obtained after loading a 1 ml affinity nickel column (GE Healthcare) with the soluble fraction of the bacterial lysate. Low molecular weight (LMW) protein standards for SDS PAGE (GE Healthcare) are shown. The expected molecular masses of 10.53 and 26.25 kDa respectively were indicated by broken lines. The proteins in the major band (indicated by an arrow) migrate with an apparent molecular mass of about 15 and 27 kDa, respectively. B, D. Elution profile of purified His-tagged VPgs from a Superdex 75 HR10/30 column (GE Healthcare) in 50 mM Tris-HCl pH 8, 300 mM NaCl, at a flow rate of 0.5 ml/min. The proteins were eluted in a major peak with an apparent molecular mass of about 17 and 40 kDa respectively as deduced from column calibration with low molecular weight protein standards for gel filtration (GE Healthcare).

Figure 2

Far UV-CD spectra of RYMV and LMV VPgs. CD spectra of purified RYMV (A) and LMV VPgs (B) in the absence (black line) or in the presence of 5% (brown line), 10% (red line), 20% (orange line) and 30% (yellow line) of TFE.

Figure 3

Disorder predictions of sobemoviral VPgs. Five predictors were used: PONDR^® VLXT, FoldIndex^©, DISOPRED2, VSL2, IUPred. The location of predicted disordered regions (in the order provided by the above-listed predictors) was schematically represented by lines along the VPg sequence. Numbering indicates the VPg length. The consensus predicted α-helices and β-strands are indicated. The sites involved in RYMV virulence (*) are indicated. The VPgs experimentally demonstrated to be disordered are shaded. RYMV Rice yellow mottle virus, CoMV Cocksfoot mottle virus, RGMoV Ryegrass mottle virus, SBMV Southern bean mosaic virus, SCPMV Southern cowpea mosaic virus, SeMV Sesbania mottle virus.

Figure 4

Disorder predictions of potyviral VPgs. Five predictors were used: PONDR^® VLXT, FoldIndex^©, DISOPRED2, VSL2, IUPred. The location of predicted disordered (in the order provided by the above-listed predictors) was schematically represented by lines along the VPg sequence. Numbering indicates the VPg length. Highly conserved regions (grey) and consensus predicted α-helices and β-strands are indicated. The conserved tyrosine (Y) involved in VPg urydylylation and the sites (*) involved in virulence are indicated. The VPgs experimentally demonstrated to be disordered are shaded. LMV Lettuce mosaic virus, PVY Potato virus Y, PVA Potato virus A, TEV Tobacco etch virus, TuMV Turnip mosaic virus, BYMV Bean yellow mosaic virus.

Figure 5

Disorder predictions of caliciviral VPgs. Five predictors were used: PONDR^® VLXT, FoldIndex^©, DISOPRED2, VSL2, IUPred. The location of predicted disordered (in the order provided by the above-listed predictors) was schematically represented by lines along the VPg sequence. Numbering represents the VPg length. The consensus predicted α-helices and β-strands are indicated. The conserved tyrosine residue (Y) involved in VPg urydylylation is indicated. RHDV Rabbit hemorrhabic disease virus (Lagovirus), VESV Vesicular exanthema of swine virus (Vesivirus), SV Man Sapporo virus Manchester virus (Sapovirus) and NV Norwalk virus (Norovirus).

Figure 6

Comparison of the PONDR^® CDF and CH-plot analyses of whole protein order-disorder via distributions of VPgs within the CH-CDF phase space. Each spot represents a single VPg whose coordinates were calculated as a distance of this protein from the boundary in the corresponding CH-plot (Y-coordinate) and an average distance of the corresponding CDF curve from the boundary (X-coordinate). The four quadrants in the plot correspond to the following predictions: (-, -) proteins predicted to be disordered by CDF, but compact by CH-plot; (-, +) proteins predicted to be disordered by both methods; (+, -) contains ordered proteins; (+, +) includes proteins predicted to be disordered by CH-plot, but ordered by the CDF analysis. Open circles correspond to caliciviral VPgs, gray circles represent sobemoviral VPgs, whereas black circles correspond to potyviral VPgs.