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...

Different models have been proposed for the nature of the potexvirus transport form that moves from cell to cell over the infected plant: (i) genomic RNA moves as native virions; or (ii) in vitro-assembled non-virion ribonucleoprotein (RNP) complexes consisting of viral RNA, coat protein (CP) and movement protein (MP), termed TGBp1, serve as the transport form in vivo. As the structure of these RNPs has not been elucidated, the products assembled in vitro from potato virus X (PVX) RNA, CP and TGBp1 were characterized. The complexes appeared as single-tailed particles (STPs) with a helical, head-like structure composed of CP subunits located at the 59-proximal region of PVX RNA; the TGBp1 was bound to the terminal CP molecules of the head. Remarkably, no particular non-virion RNP complexes were observed. These data suggest that the CP-RNA interactions resulting in head formation prevailed over TGBp1-RNA binding upon STP assembly from RNA, CP and TGBp1. STPs could be assembled from the 59 end of PVX RNA and CP in the absence of TGBp1. The translational ability of STPs was characterized in a cell-free translation system. STPs lacking TGBp1 were entirely non-translatable; however, they were rendered translatable by binding of TGBp1 to the end of the head. It is suggested that the RNA-mediated assembly of STPs proceeds via two steps. Firstly, non-translatable CP-RNA STPs are produced, due to encapsidation of the 59-terminal region. Secondly, the TGBp1 molecules bind to the end of a polar head, resulting in conversion of the STPs into a translatable form.

Previously we showed that encapsidated potato virus X (PVX) RNA is nontranslatable in vitro, but can be converted into a translatable form after binding to PVX particles of PVX-coded movement protein, the product of the first gene of triple gene block (TGBp1). Here we report that a similar effect occurs via in situ phosphorylation of the PVX coat protein (CP) by Ser/Thr protein kinase (PK) C, the mixture of casein kinases I and II or by cytoplasmic PK(s) from Nicotiana glutinosa leaves. Immunochemical analyses indicated that phosphorylation induced conformational changes in PVX CP. The N-terminal region of the PVX CP, rich in Ser and Thr residues, is exposed at the virion surface and can be removed by treatment with trypsin. We showed that (i) trypsin treatment removed the bulk of (32)P-radioactivity from in situ phosphorylated PVX CP, (ii) PVX containing N-terminally truncated CP (PVX-Ptd) failed to be translationally activated by phosphorylation, and (iii) the specific infectivity of PVX-Ptd was reduced. However, the PVX-Ptd RNA remained intact and PVX-Ptd could be translationally activated by the PVX MP TGBp1. We hypothesize that phosphorylation of the parental PVX by cytoplasmic PK(s) in vivo renders PVX RNA translatable in primary inoculated cells, whereas translational activation of the progeny virions destined for plasmodesmata trafficking is triggered by TGBp1.

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