Partially disordered structure in intravirus coat protein of potyvirus potato virus A.

Potyviruses represent the most biologically successful group of plant viruses, but to our knowledge, this work is the first detailed study of physicochemical characteristics of potyvirus virions. We measured the UV absorption, far and near UV circular dichroism spectra, intrinsic fluorescence spectra, and differential scanning calorimetry (DSC) melting curves of intact particles of a potato virus A (PVA). PVA virions proved to have a peculiar combination of physicochemical properties. The intravirus coat protein (CP) subunits were shown to contain an unusually high fraction of disordered structures, whereas PVA virions had an almost normal thermal stability. Upon heating from 20 °C to 55 °C, the fraction of disordered structures in the intravirus CP further increased, while PVA virions remained intact at up to 55 °C, after which their disruption (and DSC melting) started. We suggest that the structure of PVA virions below 55 °C is stabilized by interactions between the remaining structured segments of intravirus CP. It is not improbable that the biological efficiency of PVA relies on the disordered structure of intravirus CP

Heating-induced transition of Potyvirus Potato Virus A coat protein into β-structure

<div><p>In our previous communication, we have reported that virions of plant Potyvirus Potato Virus A (PVA) have a peculiar structure characterized by high content of disordered regions in intravirus coat protein (CP). In this report, we describe unusual properties of the PVA CP. With the help of a number of physicochemical methods, we have observed that the PVA CP just released from the virions by heating at 60–70 °C undergoes association into oligomers and transition to β- (and even cross-β-) conformation. Transition to β-structure on heating has been recently reported for a number of viral and non-viral proteins. The PVA CP isolated by LiCl method was also transformed into cross-β-structure on heating to 60 °C. Using the algorithms for protein aggregation prediction, we found that the aggregation-prone segments should be located in the central region of a PVA CP molecule. Possibly this transition mimics some functions of PVA CP in the virus life cycle in infected plants.</p></div

<i>Potato virus A</i> coat protein secondary structure prediction.

<p><i>Potato virus A</i> coat protein secondary structure prediction.</p

Characteristics of purified PVA B11 virions.

<p>(<b>A</b>) PAGE; preparations were separated by discontinuous Tris-glycine 13% SDS-PAGE. For Mw determination Page Ruler Prestained Protein ladder (Fermentas SM0671) was used for Mw determination (lane M). Purified virus aliquots of 1 µg (1) and 10 µg (2) per lane were used. (<b>B</b>) Electron microscopy of sap from PVA-infected <i>N. benthamiana</i>; magnification ×20,000.</p

Intrinsic fluorescence spectra of intact and 0.15% SDS-disrupted PVA virions.

<p>Excitation by 280 nm light at 25°C. Duration of incubation in 0.15% SDS are shown in the upper right corner. Sample concentration was 0.03 mg/ml.</p

UV absorption (A) and far UV CD (B) spectra of PVA virions.

<p>(<b>A</b>) Directly measured UV absorption spectrum of intact PVA virions in 10 mM phosphate buffer, pH 7.0 (solid line) and scattering-corrected (dotted line) spectrum are shown. (<b>B</b>) Far UV CD spectra of intact (solid line) and 0.15% SDS-disrupted (dotted line) PVA virions in 10 mM phosphate buffer were measured in 1-mm cells at 25°C at PVA concentration of 0.14 mg/ml.</p

Prediction of folded and unfolded regions in isolated PVA CP was performed using the POODLE-S, Iupred, VL3H, and FoldUnfold programs.

<p>Prediction of folded and unfolded regions in isolated PVA CP was performed using the POODLE-S, Iupred, VL3H, and FoldUnfold programs.</p

Near UV CD spectrum of intact PVA virions in 10 mM phosphate buffer, pH 7.0.

<p>Spectrum was measured at 25°C in 0.5-cm cells at PVA concentration of 0.6 mg/ml.</p

DSC melting curve for intact PVA virions in comparison with those of rod-shaped TMV virions and filamentous PVX virions in 10 mM phosphate buffer, pH 7.0.

<p>Melting temperatures (T_m,°C), enthalpy values (ΔH), and width at half height (W_hight/2 ) are shown in the upper right corner.</p

Thermal denaturation of intravirus PVA CP controlled by fluorescence (A and B) and far UV CD (C and D).

<p>Concentration and buffer are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067830#pone-0067830-g002" target="_blank">Fig.2</a>. (<b>A</b>) Temperature dependences of fluorescence maximum position (circles) and intensity (diamonds); (<b>C</b>), Temperature dependences of [θ]_203. (<b>B</b> and <b>D</b>) Complete spectra at indicated temperatures.</p