Thermal transition of native tobacco mosaic virus and RNA-free viral proteins into spherical nanoparticles

The rod-like particles of tobacco mosaic virus (TMV) of 18 nm diameter and 300 nm modal length consist of 2130 identical 17.5 kDa protein subunits arranged helically into a rigid tube. The viral RNA is intercalated between the protein turns (Zaitlin & Israel, 1975; Butler, 1999; Klug, 1999). TMV can be disassembled into protein subunits with subsequent reassembly (reconstitution) of viral particles in vitro from the nucleic acid and coat protein (CP) (Butler & Klug, 1978; Fraenkel-Conrat & Singer, 1999; Klug, 1999). In the absence of nucleic acid, the viral CP may be assembled into several types of aggregate. It has been established that polymerization of TMV CP is an endothermic, concentration-dependent and reversible process. TMV protein polymerizes when the concentration and/or temperature is increased and depolymerizes when they are decreased (Lauffer & Stevens, 1968). At a pH of approximately 6.5, TMV CP can be repolymerized into virus-like particles that are structurally similar to native virions (Anderer, 1963; Caspar, 1963; Butler & Klug, 1978; Namba et al., 1989; Butler, 1999). At a pH near 8.0 and at low ionic strength, a mixture of monomers and two-layer trimers, called A-protein, is formed (Schramm & Zillig, 1955; Lauffer & Stevens, 1968; Butler & Klug, 1978; Butler, 1999). The predominant aggregate at neutral pH and low ionic strength is a 20S two-layer polar disc made of 34 subunits (Díaz-Avalos & Caspar, 1998).

Lauffer & Price (1940) found that heat inactivation of TMV is closely associated with CP denaturation. Hart (1956) used electron microscopy to analyse the morphological changes induced in TMV by heating and reported that heating in the range 80–98 °C for 10 s resulted in a swelling of TMV particles at one or both ends. Eventually, the rods were converted into ‘ball-like particles' with the approximate volume of the original rod.

Here, we found that the size of the spherical nanoparticles (SNPs) generated by heating TMV did not necessarily correlate with that of the original rod, but varied in a wide range from approximately 50 to 800 nm. The SNPs were not only generated by the native TMV rods, but were also readily produced by different forms of RNA-free TMV CP. The evidence implied that, upon thermal denaturation, the CP subunits acquired a specific conformation favourable for assembly into SNPs of widely varying size. An additional point to emphasize is that SNPs represent a new type of particle nanoplatform capable of producing compositions of SNPs with foreign protein molecules bound to their surface.

Transmission electron microscopy (TEM) examination of the swellings revealed that the heated TMV particles underwent at least two structural transitions in the course of SNP formation. In the first stage, heating of TMV up to 90 °C was manifested as a swelling at one or both ends of the rod. Significantly, the majority of particles produced were not spherical, but represented irregular particles (IPs) of varying sizes and shapes. Numerous discrete IPs accumulated (Fig. 1a⇓). Subsequent heating of the IPs at 94 °C resulted in mature spherical SNP generation. No residual IPs were revealed after heating TMV at 94 °C, indicating that 100 % of the IPs were converted to SNPs. Heating of the native TMV in the range 94–98 °C invariably resulted in conversion of 100 % of TMV rods into SNPs (Fig. 1b⇓−d). For experimental details, see Supplementary Methods (available in JGV Online).

The IPs appeared to represent immature intermediate precursors of SNPs, suggesting that the TMV-to-SNP transition is a two-step process. The volume of the SNPs varied over a wide range and did not necessarily correspond to the volume of the individual 300 nm rods. The size of the SNPs generated following TMV heating depended heavily on virus concentration. In particular, the diameter of the SNPs obtained by heating TMV at concentrations of 0.1, 1.0 and 10.0 mg ml⁻¹ were in ranges of 50–160, 100–340 and 250–800 nm, respectively (Figs 1b⇑−d and 2⇓). The spherical shape and concentration dependence of TMV-generated SNP size was also illustrated by scanning electron microscopy (SEM) (Fig. 1e⇑−g). The SNP size dependence on TMV concentration provided evidence that both intra-particle and inter-particle interactions of thermally denatured CP subunits were involved in the TMV-to-SNP transition.

The calculated diameter of SNPs corresponding to the volume of individual TMV particles was 52.6 nm. Therefore, SNPs with a diameter close to this size were referred to as SNP monomers. The presence of SNPs smaller than 50 nm appeared to be due to the presence of fragmented TMV rods. It is reasonable to suggest that the small SNPs originated from individual TMV virions (or their fragments) fused into larger particles. Therefore, the large SNPs with diameters of 800 nm or more (Figs 1d, g⇑ and 2⇑) would correspond to a considerable number of SNP monomers. The mechanism of TMV-generated SNP fusion, if any, is not clear. Individual TMV particles might bind side by side or/and head to tail and swell together, producing SNPs of various sizes upon heating.

All types of SNP were water insoluble and could exist either as a colloidal solution or as a relatively stable suspension (depending on SNP size). We found that the SNPs were highly stable to different factors: (i) the size of SNPs remained unchanged in storage at 4 °C for at least 6 months; (ii) the SNPs did not change their size when they were precipitated by centrifugation at 10 000 g and then resuspended; (iii) the size of SNPs was not affected by repeated heating of SNP preparations up to 98 °C and cooling down; (iv) SNP sizes were unaffected by repeated freezing at −20 °C and subsequent thawing at room temperature (data not shown).

Spectrophotometry of the supernatant and SNP pellet obtained after centrifugation showed that viral RNA was completely released following SNP generation. Agarose (1 %) gel electrophoresis indicated that the released RNA was drastically degraded (data not shown). Consequently, SNPs consisted of thermally denatured CP and did not contain RNA. A single protein band with the mobility of TMV CP was revealed by SDS-PAGE of the CP from native virus and SNPs (see Supplementary Fig. S1, available in JGV Online). Therefore, no degradation (fragmentation) of TMV CP occurred upon TMV heating and SNP assembly. However, the protein isolated from SNPs by acetic acid dissociation could not be reassembled into any regular structure, indicating that the thermal denaturation was irreversible.

Initially, we thought that individual nucleoprotein TMV helical particles were essential for SNP generation. However, the data presented here showed that, in addition to native TMV, different forms of RNA-free TMV CP also produced SNPs upon heating. Four major types of TMV CP were used in this work: (i) RNA-free helical virus-like particles, (ii) double-disc aggregates, (iii) A-protein and (iv) individual CP subunits obtained at pH 13. It was found that a mixture of SNPs heterogeneous in size was produced by all four types of TMV protein upon heating. The RNA-free CP preparations appeared to be less stable than native TMV. Therefore, we checked the transition to SNPs of RNA-free CP preparations at a temperature considerably lower than 94 °C. Fig. 1(h)⇑ illustrates typical results of SNP generation by the different RNA-free TMV CP preparations at 65 °C (see Fig. 2⇑ for details). Two types of SNP were generated upon heating RNA-free CP, namely, the large particles heterogeneous in size, similar to those generated by native TMV, and many heterogeneous SNPs considerably smaller than the SNP monomer (Fig. 1h⇑). By convention, SNPs less than 35–40 nm in diameter generated by RNA-free TMV proteins were referred to as ‘mini SNPs’.

Assembly of disc-like aggregates from A-protein (at concentrations of 0.1, 1.0 and 10.0 mg ml⁻¹) was performed in 50 mM sodium phosphate buffer (pH 7.0). No SNPs were revealed after heating the disc-like aggregates up to 50 °C, whereas the mixture of large and mini SNPs was generated at all three protein concentrations following heating up to 65 °C.

It has been shown that the equilibrium between monomer subunit and cyclical trimer (A-protein) was shifted to dissociation of the 4S A-protein aggregate into 2S monomers by lowering the concentration to 1 mg ml⁻¹, increasing the pH and decreasing the ionic strength and temperature (Lauffer & Stevens, 1968). We examined the possibility of SNP generation by heating A-protein preparations at low ionic strength (10 mM Tris/HCl) and high pH (pH 7.8). Again, it was found that A-protein was capable of generating a mixture of SNPs at 65 °C (Fig. 1h⇑). Considerable amounts of mini SNPs together with large SNPs were produced. These results provided evidence that a trimer of subunits and/or individual CP subunits could be involved in SNP generation. Taken together, our data indicated that the specific helical arrangement of CP subunits is not essential for TMV CP-to-SNP transition.

It has long been known that individual CP subunits are obtained when A-protein is taken at pH 13 (Anderer, 1959; Wittmann, 1959). In order to study the possibility of CP subunit transition and assembly into SNPs, a preparation of A-protein (0.1 mg ml⁻¹) at pH 13 was heated up to 94 °C. It was found that, like the RNA-free protein aggregates described above, the TMV protein dissociated into individual subunits could be converted into a mixture of mini and large SNPs by heating. These results presented are illustrated schematically in Fig. 2⇑.

Two peculiar features distinguished SNP generation by the different forms of RNA-free CP from that of TMV: firstly, SNPs generated by RNA-free CP never contained IPs and no changes in size or shape occurred after subsequent heating up to 98 °C; secondly, the size of the SNPs generated by RNA-free CP did not depend on protein concentration. These observations provided evidence that conversion of RNA-free proteins into SNPs proceeded by a one-step assembly.

The results of an indirect ELISA showed that the SNPs were significantly more immunogenic in mice than native TMV. The specific antibody response to SNPs was up to 20 times higher than the antibody response to TMV (data not presented). Here, we showed that SNP is antigenically distantly related to native TMV (see Supplementary Fig. S2, available in JGV Online). Thereafter, the question arose as to whether SNPs generated by native TMV and those produced by other forms of TMV CP were antigenically different. It is widely known that native TMV and A-protein represent structurally and antigenically distinct CP forms. Our results demonstrated that the SNPs generated by native TMV and A-protein were antigenically closely related and morphologically similar (Fig. 1⇑).

These results allowed us to presume that a specific conformation of CP subunits favourable for SNP assembly (‘SNP-generating’ conformation) could be caused by thermal denaturation of various forms of the TMV CP. This supports the idea that a unique ‘SNP-generating’ conformation of thermally misfolded CP subunits leads to their selective assembly into SNPs.

Here, evidence has been provided that SNPs represent a new type of nanoplatform for compositions of SNPs with foreign protein molecules attached to their surface. This was tested with a recombinant GFP as a model antigen containing six His residues at the N terminus and five Arg residues at the C terminus. Native electrophoresis indicated that the SNP had a total negative charge. Therefore, five Arg residues were added to the GFP hoping to increase the total positive charge and the GFP–SNP electrostatic interaction (see Supplementary Methods).

We showed that SNPs were capable of binding the GFP molecules to their surface. Fluorescence microscopy data (Fig. 3⇓) indicated that the whole surface of all SNPs used for GFP binding was covered with fluorescent molecules. Thus, SNP can serve as a novel particle platform in nanotechnology. The SNP nanoplatform has no analogues in nature, is stable and can be used for the formation of various nanocomposites. Additional work by us (unpublished) has shown that SNPs can be used as a nanoplatform for foreign antigen/epitope presentation on their surface. Thus, this type of nanocomposite can be regarded as a candidate nanovaccine.