Stability and assembly in vitro of bacteriophage PP7 virus-like particles
© Caldeira and Peabody; licensee BioMed Central Ltd. 2007
Received: 29 June 2007
Accepted: 26 November 2007
Published: 26 November 2007
The stability of a virus-like particle (VLP) is an important consideration for its use in nanobiotechnology. The icosahedral capsid of the RNA bacteriophage PP7 is cross-linked by disulfide bonds between coat protein dimers at its 5-fold and quasi-6-fold symmetry axes. This work determined the effects of these disulfides on the VLP's thermal stability.
Measurements of the thermal denaturation behavior of PP7 VLPs in the presence and absence of a reducing agent show that disulfide cross-links substantially stabilize them against thermal denaturation. Although dimers in the capsid are linked to one another by disulfides, the two subunits of dimers themselves are held together only by non-covalent interactions. In an effort to confer even greater stability a new cross-link was introduced by genetically fusing two coat protein monomers, thus producing a "single-chain dimer" that assembles normally into a completely cross-linked VLP. However, subunit fusion failed to increase the thermal stability of the particles, even though it stabilized the isolated dimer. As a step toward gaining control of the internal composition of the capsid, conditions that promote the assembly of PP7 coat protein dimers into virus-like particles in vitro were established.
The presence of inter-dimer disulfide bonds greatly stabilizes the PP7 virus-like particle against thermal denaturation. Covalently cross-linking the subunits of the dimers themselves by genetically fusing them through a dipeptide linker sequence, offers no further stabilization of the VLP, although it does stabilize the dimer. PP7 capsids readily assemble in vitro in a reaction that requires RNA.
Viruses and VLPs are currently under investigation for a variety of uses that include confinement of chemical reactions, as templates for materials synthesis, as molecular electronics components, as platforms for polyvalent display of antigens and other ligands, and for targeted drug delivery. For some relevant examples see references [1–13]. The single-strand RNA bacteriophages offer certain advantages for such applications. VLPs can be produced in large quantities by self-assembly of a single coat protein polypeptide expressed from a plasmid, thus allowing extensive genetic manipulation of the capsid without the constraints imposed by the necessity to maintain virus viability . Engineering is further facilitated by detailed knowledge of the three-dimensional structures of RNA phages [15–22].
The physical stability of a VLP is clearly one of the factors that influence its suitability for a given application. The capsids of certain RNA phages are cross-linked by disulfide bonds between coat protein dimers at the five-fold and quasi six-fold symmetry axes, and these cross-links are expected to stabilize the capsid. It is well known that naturally occurring disulfide bonds generally stabilize protein structure (see ref , for example). The experiments reported here confirm this expectation for VLPs of the Pseudomonas RNA phage PP7.
Results and discussion
PP7's disulfide bonds stabilize the capsid
Capsid stability was assessed by measuring the quantity of intact VLP and soluble protein remaining after incubation at different temperatures (see Materials and Methods for details). Briefly, samples of purified PP7 VLPs were heated in a PCR thermocycler in either the presence or absence of dithiothreotol (DTT), and, after two minutes, the samples were chilled on ice and subjected to centrifugation at 13,000 rpm in a microcentrifuge. The pellet and supernatant were designated as insoluble and soluble fractions respectively and the amount of protein in each was determined by the assay of Bradford . The soluble fraction was also analyzed by agarose gel electrophoresis under native conditions where virus-like particles have a characteristic mobility. After staining, the quantity of capsids surviving heat treatment was determined by densitometry.
It should be noted that in each case the results obtained by following the movement of coat protein from the soluble to the insoluble fraction are similar to those obtained by measuring the disappearance of capsids, thus indicating that when capsids disaggregate, the individual coat protein subunits mostly denature concomitantly to an aggregated, insoluble form.
These results show that PP7 VLPs are substantially stabilized by the presence of its disulfide bonds. This is consistent with the well-known effects of naturally occurring disulfide bonds in many different proteins , and with the enhanced stability of bacteriophage MS2 VLPs resulting from disulfide bonds introduced at its 5-fold symmetry axes by genetic modification .
Effects of fusing subunits of the coat protein dimer
Although disulfides cross-link coat protein dimers to one another in the PP7 capsid, there exists no cross-link between the two subunits of the dimer itself. Thus, pentamers and hexamers should be the largest covalent oligomers encountered when VLPs are denatured. However, adding a covalent cross-link between the two subunits of the coat protein dimer would join all 180 subunits of the capsid into a single, giant covalent molecule with a molecular weight of about 2.5 million. Would the presence of such an additional cross-link further increase capsid stability?
The proximity within the dimer of the N-terminus of one subunit to the C-terminus of the other suggested a simple means of introducing an inter-dimer covalent bond. Duplicating the coat gene and joining the two copies together in a single reading frame fuses the C-terminus of one monomer to the N-terminus of the other. Similarly constructed single-chain dimers of MS2 coat protein have been well characterized. They retain the functional characteristics of the wild-type protein; that is, they repress translation from the replicase translational operator and assemble into apparently normal VLPs. Moreover, the tethering of MS2 coat monomers to one another greatly stabilizes the dimer against chemical denaturation and frequently reverses the destabilizing effects of amino acid substitutions and peptide insertions.
Thermal stability of 2PP7 virus-like particles
Observations of the increased stability of single-chain dimers of MS2 coat protein have already been reported. The MS2 single-chain dimer is more stable than wild-type to urea denaturation  and is more resistant to the destabilizing effects of a variety of amino acid substitutions and peptide insertions [27–29]. It was reasonable to assume that a similar stabilization would occur in the case of PP7 single-chain dimers. The fact that subunit fusion failed to additionally stabilize the VLP suggests that the stability of the dimer is not a limiting factor in the stability of the capsid. In the case of the disulfide cross-linked 2PP7 particle, VLP disappearance and protein entry into the insoluble fraction are linked events; the 2PP7 capsid is a single covalent molecule and denatures as a unit. However, when the disulfides are reduced, high temperature may cause VLP disassembly without concomitant subunit denaturation. Instead, elevation of temperature may first liberate single-chain dimers. which are substantially more stable than unfused (i.e. wild-type) dimers. Their irreversible denaturation, which is monitored by entry of the polypeptide into the insoluble fraction, occurs only at higher temperatures, thus accounting for the disparity between measurements of capsid and soluble protein loss. Alternatively, at temperatures where capsid disassembly is induced, single-chain dimers might first enter a reversibly denatured state, with irreversible denaturation and aggregation occurring only at still higher temperatures.
Assembly of PP7-like particles in vitro
Preliminary measurements of the amount of radioactive PP7 RNA present in VLPs at the highest RNA concentrations suggested an RNA to coat protein dimer ratio of about 0.3. In other words, since each dimer contains a single RNA-binding site, only about a third of the ninety sites present in the capsid were actually occupied. Apparently, the capsid is unable to enclose the quantity of RNA required to fully saturate the 90 RNA-binding sites present on its inner surface. With a length of 45 nucleotides, the RNA used in these studies was substantially larger that the minimum size (no more than 28 nucleotides) required for tight binding to PP7 coat protein. The PP7 genome itself is about 3,600 nucleotides long, so the incorporation of thirty 45-mers would not exceed the presumed packaging limit of the capsid. However, the interaction of coat protein with the translational operator concentrates the RNA at the inner surface of the capsid shell where intermolecular RNA-RNA crowding might prevent higher occupancy levels. Such crowding could also account for the relative inhibition of capsid assembly observed at high operator concentrations. However, results obtained recently with the related bacteriophage MS2 suggest an additional possibility: Binding of operator RNA may induce a coat protein dimer to adopt a conformation competent to initiate, but not to efficiently propagate capsid assembly. In other words, binding of operator RNA may be necessary to put the dimer in a state that is active for nucleation of assembly, but further addition of dimers requires that some of them be present in an RNA-free conformation . Thus the presence of excess operator RNA is inhibitory of assembly.
It is well known that naturally occurring disulfide bonds generally contribute to protein stability. The observations presented here show that the presence of disulfide bonds between coat protein dimers greatly stabilizes the PP7 virus-like particle against thermal denaturation. We sought to confer additional stability by genetically fusing the two subunits of the dimer. By thus creating a covalent cross-link between coat protein monomers, all 180 polypeptides of the VLP become cross-linked, either by disulfide bonds or by the subunit fusion. Although this manipulation stabilizes the dimer itself, it offers no further stabilization of the VLP, showing that the stability of the dimer is apparently not the limiting factor in VLP stability. PP7 capsids readily assemble in vitro in a reaction that requires RNA, raising the prospect that the interior composition of the VLP can be manipulated by specific encapsidation of foreign substances coupled to the RNA.
Proteins and recombinant DNA
The cloning, over-expression and purification of PP7 coat protein have been described in detail elsewhere. To construct the single-chain PP7 dimer, the coat sequence was amplified from pP7CT with Pfu DNA polymerase and a 3'-primer complementary to plasmid vector sequences and a 5'-primer having the sequence: 5'-CCCCCGCCGTTATGGGCAAAACCATCGTTCTTTCGGTC-3'. This introduced a Bgl I site near the 5'-end of what would be the downstream copy of the coat protein coding sequence. This was subsequently joined to a naturally occurring Bgl I site near the 3'-end of the upstream copy in pP7CT to create the junction sequence shown in Figure 3. The now duplicated sequence was cloned between Xba I and Bam HI in pET3d for over-expression in E. coli. These manipulations resulted in duplication and translational fusion of the two sequences, with the last amino acid of the upstream copy (arginine) joined to the second amino acid (serine) of the downstream copy through a two-amino acid linker (tyr-gly).
Assay for thermal stability
The thermal stabilities of virus-like particles under various conditions were determined by two methods. In the first a "melting profile" was produced by heating 25 ul samples of PP7 virus-like particles at a concentration of 1.0 mg/ml in 50 mM Tris-HCl, pH 8.5, 100 mM NaCl for 2 min. at specific temperatures. When a reaction contained DTT, it was present at a concentration of 10 mM. At the end of the incubation period, the samples were chilled on ice and then and subjected to centrifugation at 13,000 rpm in an IEC MicroMax microcentrifuge for 5 minutes. The supernatants of these samples, containing the portion of the protein that remained soluble after heat treatment, were removed to a new tube. The insoluble proteins in the pellet were redissolved in 6 M urea. Measurements of the relative quantities of soluble and insoluble protein were performed by Bradford assay . Standard curves were produced using hen lysozyme as a standard and were linear over the range of the assay. For measurement of the quantity of capsids remaining after heat treatment, soluble protein was applied to a 1% agarose gel in 40 mM Tris-acetate, pH 8.0, 2 mM EDTA, and subjected to electrophoresis. The gel was then stained with ethidium bromide and photographed under UV illumination to visualize the RNA-containing VLPs. Protein was stained with coomassie brilliant blue R250. The gel was scanned with a densitometer and the quantity of protein in individual bands was determined by comparison to a standard curve produced by applying dilutions of a known quantity of PP7 virus-like particles to the same gel. The standard curve was linear over the range employed in the assay.
The rates of denaturation were determined by incubation of proteins in 50 mM Tris-HCl, pH 8.5, 100 mM NaCl, with or without DTT at 10 mM at specified temperatures. At time points reactions were quenched on ice and then analyzed for their content of capsids and of soluble and insoluble protein as described above.
Purification of dimers
Ten milligrams of PP7 or 2PP7 VLPs purified as described previously were incubated for 60 minutes in 1 ml of 50 mM Tris-HCl, pH 8.5, 6 M urea, 10 mM DTT on ice. The resulting protein was dialyzed against 10 mM acetic acid, 50 mM NaCl (about pH 4) and then applied to a 0.9 × 45 cm column of Sephadex G75 and eluted in the same buffer. Fractions of 0.7 ml were collected. Two peaks appeared in the chromatogram. Agarose gel electrophoresis shows that the first peak is made up of VLPs that failed to disassemble. The other, eluting at fraction 20, apparently represents coat protein dimers. In a separate experiment bovine serum albumin (MW = 68,000), ovalbumin (MW = 45,000), chymotrypsinogen (MW = 25,700) and lysozyme (MW = 14,400) were applied to the column as molecular weight standards. The standard proteins yielded a linear plot of elution position versus log molecular weight. Note that BSA was omitted from this analysis because it eluted in or near the void volume. Comparison to the elution behavior of the standards indicates that the second coat protein peak has a molecular weight of about 32,000, a size roughly consistent with the predicted molecular weight of about 28,000 for the coat protein dimer. Protein from the peak fractions was used in the in vitro assembly reactions.
In vitro VLP assembly
Purified dimeric PP7 coat protein (0.1 nmol) was added to reactions containing 50 mM Tris-HCl, pH 8.5 and yeast tRNA, MS2 translational operator, or PP7 translational operator RNA in amounts varying by two-fold serial dilution from 0.1 nmol to 6.3 pmol. After 30 minutes, glycerol and bromophenol blue were added and the reactions were subjected to electrophoresis in a 1% agarose gel. RNA was visualized by staining the gels with ethidium bromide followed by photography on a UV transilluminator. MS2 and PP7 translational operator RNAs were produced by transcription in vitro as described previously . In some cases RNAs were synthesized in the presence of a 32P-labeled nucleotide and could be visualized and quantitated after exposure of the gel to a Packard Cyclone phosphorimager screen. To visualize proteins, gels were stained with coomassie brilliant blue R250.
List of abbreviations
This work was supported by NIH grant 5RO1 GM042901.
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