title: High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Centro Ricerche Casaccia, Via Anguillarese 301, I-00123, Rome, Italy. raffaele.lombardi@enea.it
authors.forename: Raffaele
authors.initials: R
authors.lastname: Lombardi
authors.forename: Patrizia
authors.initials: P
authors.lastname: Circelli
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Villani
authors.forename: Giampaolo
authors.initials: G
authors.lastname: Buriani
authors.forename: Luca
authors.initials: L
authors.lastname: Nardi
authors.forename: Valentina
authors.initials: V
authors.lastname: Coppola
authors.forename: Linda
authors.initials: L
authors.lastname: Bianco
authors.forename: Eugenio
authors.initials: E
authors.lastname: Benvenuto
authors.forename: Marcello
authors.initials: M
authors.lastname: Donini
authors.forename: Carla
authors.initials: C
authors.lastname: Marusic
mesh_terms: D002846:Chromatography, Affinity; D000090266:Chromobox Protein Homolog 5; D018506:Gene Expression Regulation, Plant; D005818:Genetic Engineering / Q000379:methods; D013058:Mass Spectrometry; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014026:Nicotiana / Q000235:genetics* / Q000821:virology; D017864:Tombusvirus / Q000235:genetics*; D014758:Viral Core Proteins / Q000235:genetics*; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: C000715594:CBX5 protein, human; D034741:RNA, Small Interfering; D014758:Viral Core Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D000090266:Chromobox Protein Homolog 5