RESUMO
The high-yield expression of a neutralizing epitope from human immunodeficiency virus type 1 (HIV-1) on the surface of a plant virus and its immunogenicity are presented. The highly conserved ELDKWA epitope from glycoprotein (gp) 41 was expressed as an N-terminal translational fusion with the potato virus X (PVX) coat protein. The resulting chimeric virus particles (CVPs), purified and used to immunize mice intraperitoneally or intranasally, were able to elicit high levels of HIV-1-specific immunoglobulin G (IgG) and IgA antibodies. Furthermore, the human immune response to CVPs was studied with severe combined immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID). hu-PBL-SCID mice immunized with CVP-pulsed autologous dendritic cells were able to mount a specific human primary antibody response against the gp41-derived epitope. Notably, sera from both normal and hu-PBL-SCID mice showed an anti-HIV-1-neutralizing activity. Thus, PVX-based CVPs carrying neutralizing epitopes can offer novel perspectives for the development of effective vaccines against HIV and, more generally, for the design of new vaccination strategies in humans.
Assuntos
Vacinas contra a AIDS/imunologia , Proteínas do Capsídeo , Capsídeo/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Animais , Capsídeo/genética , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Feminino , Engenharia Genética , Vetores Genéticos , Proteína gp41 do Envelope de HIV/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Dados de Sequência Molecular , Potexvirus/genética , Potexvirus/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , VírionRESUMO
Ripe tomato fruits accumulate large amounts of the red linear carotene, lycopene (a dietary antioxidant) and small amounts of its orange cyclisation product, beta-carotene (pro-vitamin A). Lycopene is transformed into beta-carotene by the action of lycopene beta-cyclase (beta-Lcy). We introduced, via Agrobacterium-mediated transformation, DNA constructs aimed at up-regulating (OE construct) or down-regulating (AS construct) the expression of the beta-Lcy gene in a fruit-specific fashion. Three transformants containing the OE construct show a significant increase in fruit beta-carotene content. The fruits from these plants display different colour phenotypes, from orange to orange-red, depending on the lycopene/beta-carotene ratio. Fruits from AS transformants show up to 50% inhibition of beta-Lcy expression, accompanied by a slight increase in lycopene content. Leaf carotenoid composition is unaltered in all transformants. In most transformants, an increase in total carotenoid content is observed with respect to the parental line. This increase occurs in the absence of major variations in the expression of endogenous carotenoid genes.
Assuntos
Carotenoides/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , beta Caroteno/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Sequência de Bases , Primers do DNA/genética , DNA Antissenso/genética , Expressão Gênica , Genes de Plantas , Engenharia Genética , Liases Intramoleculares/genética , Liases Intramoleculares/metabolismo , Licopeno , Fenótipo , Plantas Geneticamente Modificadas , Transformação GenéticaRESUMO
Mutagenesis of the artichoke mottled crinkle virus (AMCV) genome and complementation studies between replication-defective mutants were undertaken to identify viral protein(s) essential for AMCV replication. Inoculation of Nicotiana benthamiana protoplasts with mutant transcripts revealed that null mutations in ORFs 1 [tA33(-)], 2 [tA92(-)] and 6 [tA7(-)], as well as an ORF 2 mutation [tA92GED] in the GDD motif of the 92 kDa protein, the putative replicase, prevented accumulation of detectable levels of progeny RNA. Conversely, mutations of ORFs 3 [tA41(-)], 4 [tA21(-)] and 5 [tA19(-)] did not substantially affect the accumulation of AMCV genomic and subgenomic RNAs of both positive and negative polarity. Inoculation of N. benthamiana plants with transcripts impaired in replication revealed that tA92(-) and tA7(-) mutants lead to replicating pseudorevertants. Functional analysis of these pseudorevertants showed that: (i) the double stop codon introduced at the end of ORF 1 to prevent the translational readthrough of the 92 kDa protein reverted to a single amber, ochre or opal codon, giving rise to viable genomes; (ii) the putative 7 kDa protein is not essential for genome viability, although the RNA region spanning ORF 6 plays a role in cis in replication. Finally, the two replication-defective mutants tA33(-) and tA92(-) complemented when co-inoculated to N. benthamiana protoplasts, definitively proving that the 33 kDa protein is essential for tombusvirus genome replication. Analysis of viral RNAs from the coinfection experiments showed that tA92(-) was preferentially amplified over tA33(-).
