Transgenic tobacco plants expressing either a full-length form of the tobacco etch virus (TEV) coat protein or a form truncated at the N terminus of the TEV coat protein were initially susceptible to TEV infection, and typical systemic symptoms developed. However, 3 to 5 weeks after a TEV infection was established, transgenic plants "recovered" from the TEV infection, and new stem and leaf tissue emerged symptom and virus free. A TEV-resistant state was induced in the recovered tissue. The resistance was virus specific. Recovered plant tissue could not be infected with TEV, but was susceptible to the closely related virus, potato virus Y. The resistance phenotype was functional at the single-cell level because protoplasts from recovered transgenic tissue did not support TEV replication. Surprisingly, steady state transgene mRNA levels in recovered tissue were 12-to 22-fold less than transgene mRNA levels in uninoculated transgenic tissue of the same developmental stage. However, nuclear run-off studies suggested that transgene transcription rates in recovered and uninoculated plants were similar. We propose that the resistant state and reduced steady state levels of transgene transcript accumulation are mediated at the cellular level by a cytoplasmic activity that targets specific RNA sequences for inactivation.
Transient expression is a rapid, useful approach for producing proteins of interest in plants. Tobacco mosaic virus (TMV)-based transient expression vectors can express very high levels of foreign proteins in plants. However, TMV vectors are, in general, not efficiently delivered to plant cells by agroinfection. It was determined that agroinfection was very efficient with a 35S promoterdriven TMVreplicon that lacked the TMV coat protein gene sequence. This coat protein deletion vector had several useful features as a transient expression system, including improved ease of use, higher protein expression rates, and improved biocontainment. Using this TMV expression vector, some foreign proteins were expressed at levels of 3 to 5 mg/g fresh weight of plant tissue. It is proposed that this new transient expression vector will be a useful tool for expressing recombinant proteins in plants for either research or production purposes.
Transgenic tobacco plants expressing either a full-length form of the tobacco etch virus (TEV) coat protein or a form truncated at the N terminus of the TEV coat protein were initially susceptible to TEV infection, and typlcal systemic symptoms developed. However, 3 to 5 weeks after a TEV infection was established, transgenlc plants "recovered" from the TEV infection, and new stem and leaf tissue emerged symptom and virus free. A TEV-resistant state was induced ln the recovered tissue. The resistance was virus speclflc. Recovered plant tissue could not be lnfected wlth TEV, but was susceptlble to the closely related virus, potato virus Y. The resistance phenotype was functional at the slngle-cell leve1 because protoplasts from recovered transgenic tissue did not support TEV repllcation. Surprlslngly, steady state transgene mRNA levels in recovered tissue were 12-to 22-fold less than transgene mRNA levels in uninoculated transgenic tissue of the same developmental stage. However, nuclear run-off studles suggested that transgene transcription rates in recovered and uninoculated plants were similar. We propose that the resistant state and reduced steady state levels of transgene transcript accumulation are mediated at the cellular leve1 by a cytoplasmic actlvity that targets speclfic RNA sequences for inactlvation.
Historically, the study of plant viruses has contributed greatly to the elucidation of eukaryotic biology. Recently, concurrent with the development of viruses into expression vectors, the biotechnology industry has developed an increasing number of disease therapies utilizing recombinant proteins. Plant virus vectors are viewed as a viable option for recombinant protein production. Employing pathogens in the process of creating added value to agriculture is, in effect, making an ally from an enemy. This review discusses the development and use of viruses as expression vectors, with special emphasis on (+) strand RNA virus systems. Further, the use of virus expression vectors in large-scale agricultural settings to produce recombinant proteins is described, and the technical challenges that need to be addressed by agriculturists and molecular virologists to fully realize the potential of this latest evolution of plant science are outlined.
Display of peptides or proteins in an ordered, repetitive array, such as on the surface of a virus-like particle, is known to induce an enhanced immune response relative to vaccination with the "free" protein antigen. The coat protein of Tobacco mosaic virus (TMV) can accommodate short peptide insertions into the primary sequence, but the display of larger protein moieties as genetic fusions to the capsid protein has not been possible. We employed a randomized library approach to introduce a reactive lysine at the externally located amino terminus of the coat protein, which facilitated biotinylation of the capsid. To characterize display of heterologous proteins on the virion surface, we bound a model antigen (green fluorescent protein (GFP)-streptavidin (SA), expressed and purified from plants) to the biotinylated TMV particles, creating a GFP-SA decorated virus particle. A GFP-SA tetramer loading of 26% was obtained, corresponding to approximately 2200 GFP moieties displayed per intact virion. We evaluated the immunogenicity of GFP decorated virions in both mice and guinea pigs and found augmented humoral IgG titers in both species, relative to unbound GFP-SA tetramer. Next, we fused an N-terminal fragment of the Canine oral papillomavirus L2 protein to streptavidin. With TMV display, the L2 protein fragment was significantly more immunogenic than uncoupled antigen when tested in mice. By demonstrating the presentation of whole proteins, this study expands the utility of TMV as a vaccine scaffold beyond that which is possible by genetic manipulation.
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