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

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

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

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

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

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