(on the lower leaves only, viral disease symptoms could be seen in the upper noninfiltrated leaves
(on the lower leaves only, viral disease symptoms could be seen in the upper noninfiltrated leaves. and very high accumulation titers, plant tobamoviruses provide an extremely cheap source of protein biopolymers of discrete nanoparticle sizes than can be manufactured rapidly and under very simple conditions. Tobacco mosaic virus (TMV), the best-known member of the tobamoviruses, is also one of the most extensively studied viruses (1). It has a positive-sense RNA genome encoded in a single 6.4-kb RNA molecule. The genome encodes four proteins, including the 17.5-kDa coat protein (CP), the most abundant viral product and the only component of the TMV capsid. More than 2,100 copies of CP fully protect the single-stranded viral RNA, resulting in rigid rod-shaped viral particles 300 nm in length and 18 nm in diameter, with a molecular mass of 40,000 kDa. Considering that TMV accumulates to levels of up to 10 g per kg of leaf biomass and that CP accounts for 95% of the mass of the viral particles, the CP therefore represents the most abundant protein that can be harvested from plants. The viral particles can be purified industrially by using simple low-tech protocols (1, 2). Because of the ability of the CP to polymerize and and the high stability and defined size of the assembled virions, the CP represents a potentially promising biopolymer feedstock for a number Capreomycin Sulfate of applications in nanobiotechnology. The CP-based polymer can be modified in two different ways. One approach consists of adding novel moieties to viral particles by chemical modification (3, 4). The other avenue consists of genetically reprogramming the virus to express protein fusions, thus allowing the addition of new functional blocks to the viral core monomer, the CP (5). Because the structure of the TMV particle has been determined at atomic resolution (6, 7), fusion proteins can be designed in such a way that the added sequences are predicted to lie on the surface of the assembled viral particle. Despite this knowledge, attempts by numerous groups to create new products Capreomycin Sulfate based on protein fusions have so far met with limited success. CP fusion proteins carrying an up to 20-aa extension to the C-terminus or a 12-aa insertion in a surface loop of the CP were shown to assemble into infectious virions, with the displayed peptides ranging from neuropeptides (5, 8), to vaccine epitopes (9C11), to affinity tags (12). However, addition of larger peptides at the C-terminus of TMV CP always proved to be assembly-defective. We have decided to reexamine the issue of the size constraint on proteins that can be fused to the CP of tobamoviruses without preventing viral particle formation. We have explored approaches taken by others who have designed systems for display of foreign proteins on the surface of filamentous bacteriophages (13). Among other parameters, we have tested the effect of short linkers introduced between the Rabbit polyclonal to ABHD3 two fusion components on the functionality of both fusion partners (14, 15). We report here data on the fusion of the CP of turnip vein clearing virus (TVCV) with a functional fragment of protein A from leaves by using the proviral Capreomycin Sulfate vector system described earlier (18). The CP and protein A domains were cloned separately in 5- and 3-provector modules, and these modules assembled into a complete vector by site-specific recombination, plants. Wild-type CP is expressed from an assembled vector (pICH17501). CP-protein A fusions are expressed from separate 5 and 3 modules that are assembled by using a site-specific recombinase (pICH10881). Short 15-aa linkers (hatched boxes) are included in the 5 modules (except for pICH20697): a flexible linker (GGGGS)3 in pICH20701 and pICH24384 and a helical linker (EAAAK)3 in pICH20723 and pICH24399. White boxes represent introns. RdRp, RNA-dependent RNA polymerase; MP, movement protein; attP/attB, recombination sites; int, 5 and 3 part of intron for removal of the recombination site through Capreomycin Sulfate splicing; N, 3 nontranslated region; T, NOS terminator; LB and RB, T-DNA.