Crystallization, Reentrant Melting, and Resolubilization of Virus Nanoparticles

Crystallization is a fundamental and ubiquitous process that is well understood in the case of atoms or small molecules, but its outcome is still hard to predict in the case of nanoparticles or macromolecular complexes. Controlling the organization of virus nanoparticles into a variety of 3D supramolecular architectures is often done by multivalent ions and is of great interest for biomedical applications such as drug or gene delivery and biosensing, as well as for bionanomaterials and catalysis. In this paper, we show that slow dialysis, over several hours, of wild-type Simian Virus 40 (wt SV40) nanoparticle solution against salt solutions containing MgCl₂, with or without added NaCl, results in wt SV40 nanoparticles arranged in a body cubic center crystal structure with <i>Im</i>3<i>m</i> space group, as a thermodynamic product, in coexistence with soluble wt SV40 nanoparticles. The nanoparticle crystals formed above a critical MgCl₂ concentrations. Reentrant melting and resolubilization of the virus nanoparticles took place when the MgCl₂ concentrations passed a second threshold. Using synchrotron solution X-ray scattering we determined the structures and the mass fraction of the soluble and crystal phases as a function of MgCl₂ and NaCl concentrations. A thermodynamic model, which balances the chemical potentials of the Mg²⁺ ions in each of the possible states, explains our observations. The model reveals the mechanism of both the crystallization and the reentrant melting and resolubilization and shows that counterion entropy is the main driving force for both processes

Scaffold Properties Are a Key Determinant of the Size and Shape of Self-Assembled Virus-Derived Particles

Controlling the geometry of self-assembly will enable a greater diversity of nanoparticles than now available. Viral capsid proteins, one starting point for investigating self-assembly, have evolved to form regular particles. The polyomavirus SV40 assembles from pentameric subunits and can encapsidate anionic cargos. On short ssRNA (≤814 nt), SV40 pentamers form 22 nm diameter capsids. On RNA too long to fit a <i>T</i> = 1 particle, pentamers forms strings of 22 nm particles and heterogeneous particles of 29–40 nm diameter. However, on dsDNA SV40 forms 50 nm particles composed of 72 pentamers. A 7.2-Å resolution cryo-EM image reconstruction of 22 nm particles shows that they are built of 12 pentamers arranged with <i>T</i> = 1 icosahedral symmetry. At 3-fold vertices, pentamers each contribute to a three-helix triangle. This geometry of interaction is not seen in crystal structures of <i>T</i> = 7 viruses and provides a structural basis for the smaller capsids. We propose that the heterogeneous particles are actually mosaics formed by combining different geometries of interaction from <i>T</i> = 1 capsids and virions. Assembly can be trapped in novel conformations because SV40 interpentamer contacts are relatively strong. The implication is that by virtue of their large catalog of interactions, SV40 pentamers have the ability to self-assemble on and conform to a broad range of shapes

High levels of mRNA were detected in the lungs of 2CLP animals (plasmid pGL3 used as positive control for luciferase)

<p><b>Copyright information:</b></p><p>Taken from "Simian virus 40 vectors for pulmonary gene therapy"</p><p>http://respiratory-research.com/content/8/1/74</p><p>Respiratory Research 2007;8(1):74-74.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2238754.</p><p></p> Minimal levels were present in the liver, kidney spleen and none in the heart. Minimal expression in the lungs, kidney, liver, spleen, and heart of T0 and SO animals. GAPDH was used as a house keeping control gene, using PCR primers specific for GAPDH

Parameters affecting the in vitro packaging reaction.

<p>Effect of temperature (A) and concentration of monovalent salts (B) present during the re-association reaction (step B) on the yield, measured as titer of luc transducing units.</p

Analysis of the particles.

<p>A,B-Proteins were separated by elecrtrophoresis in Tricine buffer on 16% polyacrylamide gel. Western blotting was performed with polyclonal antibody against histone H3 (Upstate) (A) and against VP1 (B). M–size marker; 1–Nuclear extracts used in the packaging reaction; 2–In vitro assembled particles, fraction 9 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000765#pone-0000765-g004" target="_blank">Fig. 4</a>. 3–wild type SV40. C-Analysis of the packaged DNA. Particles purified by ultrafiltration were treated with Tris base (200 mM) in presence of 25 mM EGTA and 25 mM DTT for 1 hr at 37°C. DNA was extracted by phenol-chloroform treatment in presence of 1% SDS, and analyzed by Southern blotting with pGL3-control as a probe.</p

Equilibrium sedimentation in CsCl gradient.

<p>The reaction products, 2 ml, were fractionated on a CsCl density gradient in stabilization buffer at pH 5.2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000765#s4" target="_blank">Materials and Methods</a>). VP1 was analyzed by Coomassie and Bradford, DNA by real-time quantitative PCR and <i>luc</i> TU as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000765#s4" target="_blank">Materials and Methods</a>.</p

A model for the <i>in vitro</i> assembly reaction.

<p>The addition of DTT to nuclear extracts (A) leads to disassembly of the VLPs. Following the addition of supercoiled DNA (B) pentamers bind along each DNA molecule. This increases the local concentration of VP1, facilitating concerted assembly. Reassembly may be facilitated by presence of chaperones in the nuclear extracts. Assembly is accompanied by DNA condensation, presumably via the action of topo II. In Step C the capsids are stabilized at pH 5.2.</p

Cooperativity of VP1 in the reaction.

<p>Effect of substrate concentration on the reaction yield. A–DNA; B–VP1. The amounts used per reaction, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000765#s4" target="_blank">Materials and Methods</a>, is indicated. C-Fourteen different batches of nuclear extracts were assayed for packaging activity. Only 11 distinct data points are seen because of overlap of some of the data points. D-Calculation of Hill coefficient from the same set of data as in C. Y is the fraction of fully assembled VP1, measured as TU.</p

The reaction requires cellular factors.

<p>A–VLPs were purified on sucrose gradient from nuclear extracts harvested on day 3. M-molecular weight marker; VLPs–purified VLPs used in the packaging experiment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000765#pone-0000765-t001" target="_blank">Table 1</a>). B-Nuclear extracts were harvested from infected Sf9 cells at different time points after infection, as designated on top, and analyzed by SDS-PAGE and Coomassie-blue staining. M-molecular weight marker; Mock-nuclear extract of Sf9 cells infected with wild type baculovirus; SV40-nuclear extract of CV1 cells infected with wild type SV40; BSA-1 µg BSA, a standard for band intensity. C-Activity of the nuclear extracts shown in part B. ○-Packaging activity measured as titer (left ordinate); □-VP1 level estimated from A (right ordinate).</p

Composition of the packaging reaction.

a<p>Final concentration of the reagents in the reaction mixture at each step is shown in parentheses.</p>b<p>Average±S.E. The titer is shown as luciferase transduction units. The number in parenthesis represents the number of repeated experiments.</p