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

Abstract

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...

Thermal conversion of filamentous potato virus X into spherical particles with different properties from virions

“…Similar results have been obtained for 48-nm particles (data not shown). TMV SPs were only distantly antigenically related to native TMV, and about 3-5% of native TMV epitopes were retained at the TMV SPs' surface [1].…”

“…Analysis of RNA in 1% agarose gel was carried out according to Karpova et al [22]; analysis of proteins was performed in SDS/PAGE (8-20%) as described by Atabekov et al [1].…”

“…The antigenic properties of native PVX and PVX SPs (mean diameter of 77 nm) were compared by conventional triple-antibody sandwich ELISA (TAS-ELISA) using rabbit IgG specific to PVX or PVX SPs as capture antibodies, polyclonal mice antisera to the same antigens and normal mouse serum (negative control) as described Atabekov et al [1].…”

Obtaining and characterization of spherical particles—new biogenic platforms

“…Продемонстрировано, что они обладают высокой электронной плотностью. Ана-лиз ультратонких срезов показал, что сферические частицы не содержат внутренних полостей и являются внутри однородными.Ключевые слова: вирусы растений, вирус табачной мозаики, сферические частицы.Вирусы растений и их вирусоподобные частицы применяют в различных областях биотехнологии: разработка вакцин и адъювантов, создание биоло-гически активных комплексов, разработка контрас-тирующих агентов, адресная доставка, микроэлектро-ника [1][2][3][4][5][6][7][8][9][10][11][12]. Важное преимущество фитовирусов состоит в том, что они непатогенны для человека [1].…”

“…Ранее было показано, что при нагревании па-лочковидных вирионов вируса табачной мозаики (ВТМ) до 94°С происходит структурная перестройка вирусного белка оболочки (БО) и формируются сферические частицы (СЧ) контролируемого раз-мера. СЧ обладают высокой стабильностью, не со-держат РНК, биодеградируемы [11][12]. Продемон-стрированы уникальные адсорбционные свойства СЧ, на их основе созданы комплексы как с раз-личными белками, так и с икосаэдрическими ви-рионами [2][3][4].…”