14 research outputs found
High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly
SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility
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-Ã… 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
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
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
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
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
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