Spherical nanoparticles (SNPs) were generated by two-step thermal remodelling of native tobacco mosaic virus (TMV) at 94 6C. Particles of irregular shape and varying size were generated by TMV at 90 6C. They could be converted into SNPs by heating at 94 6C and were considered to be intermediate precursors of SNPs. In addition to SNP monomers (53 nm diameter), generated by individual TMV virions, large SNPs (100-800 nm diameter) were assembled. The size of the SNPs depended on the TMV concentration. The SNPs could be generated by distinct forms of RNA-free TMV coat protein (CP) aggregates and individual CP subunits. A one-step SNP assembly appeared to occur in these cases. These results show that SNPs represent a new type of particle nanoplatform for producing compositions of SNPs with foreign protein molecules bound to their surface.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 u 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 eviden...

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We reported recently that RNA-free spherical particles (SPs) generated by thermal remodelling of tobacco mosaic virus (TMV) are capable of binding GFP to their surface. Here, we show that SPs represent a universal particle platform that can form compositions by binding a diversity of various foreign proteins/epitopes of viral and non-viral origin to their surface. Numerous molecules of a foreign protein linked to the SP surface were revealed by immunogold electron microscopy. Several SP-based compositions were obtained containing one of the following foreign antigens: antigenic determinant A of rubella virus E1 glycoprotein; a recombinant protein containing the M2e epitope of influenza virus A protein M2; a recombinant antigen consisting of three epitopes of influenza virus A haemagglutinin; potato virus X (PVX) coat protein (CP); BSA; and PVX CP fused with the epitope of plum pox virus CP. The 'mixed' compositions could be also assembled by binding two different foreign antigens to each of the SPs. Immunogenicity of foreign antigens adsorbed or linked covalently to SPs in the SP-based compositions was examined. The antigenic specificity of foreign antigens was retained, whereas their immunogenicity increased significantly. It was inferred that SPs exhibit immunopotentiating activity, in particular in the form of compositions comprising SP and foreign antigen linked covalently to their surface by formaldehyde.

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A new isolate of Alternanthera mosaic virus (AltMV-MU) was purified from Portulaca grandiflora plants. It has been shown that the AltMV-MU coat protein (CP) can be efficiently reassembled in vitro under different conditions into helical RNA-free virus-like particles (VLPs) antigenically related to native virus. The AltMV-MU and VLPs were examined by atomic force and transmission electron microscopies. The encapsidated AltMV-MU RNA is nontranslatable in vitro. However, it can be translationally activated by CP phosphorylation or by binding to the TGB1protein from the virus-coded movement triple gene block.