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High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

TitleHigh-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus
Publication TypeArticolo su Rivista peer-reviewed
Year of Publication2009
AuthorsLombardi, R., Circelli P., Villani Maria Elena, Buriani G., Nardi Luca, Coppola V., Bianco L., Benvenuto Eugenio, Donini Marcello, and Marusic Carla
JournalBMC Biotechnology
KeywordsA plants, Affinity, Affinity chromatography, Affinity purification, Agroinfiltration, Artichoke, artichoke mottled crinckle virus, artichoke mottled crinkle virus, article, biosynthesis, Chromatographic analysis, Chromatography, controlled study, core protein, Cynara scolymus, Expression levels, Fresh tissues, gene expression regulation, gene silencing, Genetic engineering, genetics, High yield, HIV-1 Nef, Human immunodeficiency virus, Human immunodeficiency virus 1, Immobilized metal ion affinity chromatographies, In-plants, In-vacuum, Ion chromatography, isolation and purification, Low-yield, Mass Spectrometry, Metal ions, Metal refining, methodology, molecular cloning, Multicomponents, nef Gene Products, Nef protein, Nicotiana benthamiana, nonhuman, Nucleic acids, nucleotide sequence, Plant, plant virus, Plants (botany), Potato virus, Potato virus X, protein expression, protein p19, protein purification, Proteins, Purification, Purification protocol, RNA, RNA Interference, RNA silencing, Small Interfering, Small interfering RNA, Stable transformation, Tobacco, Tomato bushy stunt virus, Tombusvirus, Total soluble protein, transgene, transgenic plants, transient expression, vacuum, Viral Core Proteins, Viral infections, Virology, Viruses

Background: In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results: The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion: We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine. © 2009 Lombardi et al; licensee BioMed Central Ltd.


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Citation KeyLombardi2009