Abstract
BackgroundIn 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.ResultsThe 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.ConclusionWe 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.
Highlights
In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems
Cloning of the AMCV-P19 gene silencing suppressor AMCV viral cDNA was retrotranscribed from genomic RNA extracted from purified virus particles as previously described [25]
The amcv-p19 gene was amplified from viral cDNA by polymerase chain reaction (PCR), using specific primers flanking the p19 coding region [26]
Summary
Different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. The major advantages of plants over traditional expression systems based on bacterial and mammalian cells are a reduced risk of contamination by human pathogens and low costs, especially for large-scale production [2]. It has been demonstrated that both structural and regulatory proteins of HIV or related viruses can be successfully expressed in plants by either stable transformation (nuclear or plastid transformation) or transient expression systems (plant virus vectors and leaf agroinfiltration) [3]. The non-structural HIV-1 accessory protein Nef is considered a good candidate for the formulation of vaccines that combine both structural and functional viral components. The effects of a genetic vaccine combining both structural (Gag/Pol, Env) and regulatory (Rev, Tat, Nef) viral proteins were evaluated in the SIVMacaca animal model [10,11]
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