Isolated Potato Virus A coat protein possesses unusual properties and forms different short virus-like particles

<p>In our previous study, we have observed that the isolated coat proteins (CP) of the Potyvirus Potato Virus A (PVA) virions exhibit an intrinsic tendency to self-associate into various multimeric forms containing some fractions of cross-β-structure. In this report, we studied the effect of solution conditions on the structure and dissociation of isolated PVA CP using a number of complementary physicochemical methods. Analysis of the structure of PVA CP in solution was performed by limited proteolysis with MALDI-TOF mass spectrometry analysis, transmission electron microscopy, intrinsic fluorescence spectroscopy, and synchrotron small angle X-ray scattering (SAXS). Overall structural characteristics of PVA CP obtained by combination of these methods and <i>ab initio</i> shape reconstruction by SAXS show that PVA CP forms large multi-subunit particles. We demonstrate that a mixture of compact virus-like particles (VLP) longer than 30 nm is assembled on dialysis of isolated CP into neutral pH buffer (at low ionic strength). Under conditions of high ionic strength (0.5 M NaCl) and high pH (pH 10.5), PVA dissociates into low compactness oval-shaped particles of approximately 30 subunits (20–30 nm). The results of limited trypsinolysis of these particles (enzyme/substrate ratio 1:100, 30 min) showed the existence of non-cleavable core-fragment, consisting of 137 amino acid residues. Trypsin treatment removed only a short N-terminal fragment in the intact virions. These particles are readily reassembled into regular VLPs by changing pH back to neutral. It is possible that these particles may represent some kind of intermediate in PVA assembly <i>in vitro</i> and <i>in vivo</i>.</p

Oligomeric species of GadA.

<p>GadA consists of hexamers and dimers in solution that are structurally sensitive to sample conditions. <b>A.</b> SASREFMX models of the GadA hexamer and associated dimes from samples stored in higher ionic strength conditions. <b>B.</b> SASREFMX models of the GadA hexamer and dimer in low-salt conditions. <b>C.</b> Spatial overlay of the GadA crystal structure (red) with the GadA hexamer from the hexamer-dimer mixture obtained in low-salt conditions. The associated fits of the mixtures are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156105#pone.0156105.g002" target="_blank">Fig 2B and 2C</a>.</p

Shape restoration of FbaB and PPase by <i>ab initio</i> and rigid body methods.

<p><b>A.</b> A DAMMIN <i>ab initio</i> model of the FbaB decamer (light grey spheres, P52 symmetry) superimposed with a rigid body model showing the arrangement of the individual protomers (represented as different colored ribbons). Two orientations are shown. <b>B.</b> A comparison of the subunit orientation of PPase from the X-ray crystal structure (i) compared to the rigid body model (ii) determined from SAXS data. A single protomer has been colored green to highlight the apparent rearrangement of the PPase subunits that occurs in solution. <b>C.</b> The DAMMIN <i>ab initio</i> bead model of the PPase hexamer (light grey spheres/surface; P32 symmetry) superimposed with the SASREF rigid-body model of the enzyme (each protomer of the PPase hexamer are shown as different colored ribbons).</p

Superposition of the active site residues of Class I Fba.

<p>The predicted active site topology of the <i>E</i>. <i>coli</i> enzyme (derived from I-Tasser, Model 1) is shown in dark green on both figure panels. <b>A.</b> Spatial alignment of <i>E</i>. <i>coli</i> FbaB and the X-ray crystal structure of FbaB from <i>Thermoproteus tenax</i> (PDB: 1W8R [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156105#pone.0156105.ref052" target="_blank">52</a>]). <b>B.</b> The alignment between <i>E</i>. <i>coli</i> FbaB relative to <i>Oryctolagus cuniculus</i> FbAB (PDB: 1ZAI [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156105#pone.0156105.ref009" target="_blank">9</a>]). The substrate fructose-1,6-bisphosphate (FBP), taken from the 1W8R crystal structure, is represented as a ball-and-stick in panel A. The amino acid numbers of the corresponding proteins are given in black for the X-ray crystal structures and in green for the I-Tasser <i>E</i>.<i>coli</i> model.</p

Oligomeric species of KduI.

<p>KduI consists of hexamers (i), dimers (ii), as well as extended (iii) and stacked dodecamers (iv). The corresponding fit to the SAXS data of the mixture is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156105#pone.0156105.g003" target="_blank">Fig 3A</a>, curve 3 (hexamers, <i>v</i>_<i>i</i> = 0.31; stacked dodecamers, <i>v</i>_<i>i</i> = 0.17; extended dodecamers, <i>v</i>_<i>i</i> = 0.24 and; dimers, <i>v</i>_<i>i</i> = 0.28).</p

Guinier and Kratky plots for PPase, FbaB, KduI and GadA.

<p><b>A.</b> Guinier plots for (1) FbaB and (2) PPase. <b>B.</b> The corresponding Kratky plots for FbaB (1) and PPase (2). <b>C.</b> Guinier plots for KduI (1) showing a double-slope Guinier region (corresponding to <i>R</i>_<i>g</i> = 6.2 nm and <i>R</i>_<i>g</i> = 4.5 nm). Plots (2) and (3) show the results obtained from the GadA and GadA low-salt samples. <b>D.</b> The corresponding Kratky plots of KduI (1), GadA (2) and GadA low-salt (3).</p

Overall fold of Class I Fba from <i>E</i>. <i>coli</i> as predicted using secondary and tertiary structure modeling.

<p><b>A.</b> A profile view of FbaB Model 1 and; <b>B.</b> A top view along the axis of the TIM-barrel fold. The β-strands and α-helices of the TIM barrel are colored gold and dark cyan, respectively. Light green α-helices are not involved in forming the TIM-barrel core motif.</p

Scattering data from KduI (A) and GadA (B) and GadA low-salt (C).

<p>In all panels, (1) represents experimental data and (2) the scattering-fit computed from the respective hexameric X-ray crystal structures of either KduI or GadA. OLIGOMER fits to the data for the respective mixtures are also displayed. For KduI, curve (3)—panel A—shows the fit of the hexamer, dimer and dodecamer mixture of the models presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156105#pone.0156105.g007" target="_blank">Fig 7</a>. For the GadA and GadA low-salt samples, curve (3) shows the OLIGOMER fit of the crystallographic hexamer and all associated crystallographic dimers, while curve (4) shows the best fitting hexamer-dimer mixtures modelled using SASREFMX as displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156105#pone.0156105.g008" target="_blank">Fig 8A and 8B</a>, respectively. Note: for visualization purposes, the identical SAXS profile for KduI (4 mg/ml), GadA (8.5 mg/ml) and GadA low-salt (5 mg/ml) have been duplicated and shifted on the <i>I</i>(<i>s</i>) axis.</p

Overall structural parameters of the proteins.^a

<p>Overall structural parameters of the proteins.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156105#t001fn001" target="_blank">^a</a></p

Structural Analysis of Influenza A Virus Matrix Protein M1 and Its Self-Assemblies at Low pH

<div><p>Influenza A virus matrix protein M1 is one of the most important and abundant proteins in the virus particles broadly involved in essential processes of the viral life cycle. The absence of high-resolution data on the full-length M1 makes the structural investigation of the intact protein particularly important. We employed synchrotron small-angle X-ray scattering (SAXS), analytical ultracentrifugation and atomic force microscopy (AFM) to study the structure of M1 at acidic pH. The low-resolution structural models built from the SAXS data reveal a structurally anisotropic M1 molecule consisting of a compact NM-fragment and an extended and partially flexible C-terminal domain. The M1 monomers co-exist in solution with a small fraction of large clusters that have a layered architecture similar to that observed in the authentic influenza virions. AFM analysis on a lipid-like negatively charged surface reveals that M1 forms ordered stripes correlating well with the clusters observed by SAXS. The free NM-domain is monomeric in acidic solution with the overall structure similar to that observed in previously determined crystal structures. The NM-domain does not spontaneously self assemble supporting the key role of the C-terminus of M1 in the formation of supramolecular structures. Our results suggest that the flexibility of the C-terminus is an essential feature, which may be responsible for the multi-functionality of the entire protein. In particular, this flexibility could allow M1 to structurally organise the viral membrane to maintain the integrity and the shape of the intact influenza virus.</p></div