RESUMEN
The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.
Asunto(s)
Endopeptidasas/química , Endopeptidasas/aislamiento & purificación , Virus del Mosaico/enzimología , Proteínas Virales/química , Proteínas Virales/aislamiento & purificación , Secuencia de Aminoácidos , ADN Complementario/química , ADN Complementario/metabolismo , ADN Viral/química , ADN Viral/metabolismo , Elettaria/virología , Endopeptidasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Virus del Mosaico/metabolismo , Especificidad por Sustrato , Proteínas Virales/metabolismoRESUMEN
Bhendi yellow vein mosaic disease (BYVMD) is caused by the association of a DNA beta satellite with a begomovirus component. The begomovirus component has two promoters, one in the virion sense (V-sense) and the other in the complementary sense (C-sense) in the intergenic region (IR). To study the promoter activities of V-sense and C-sense promoters, mGFP gene fusion was made downstream to the promoters. Transient and stable expressions in N. benthamiana leaves showed significant GFP expression under C-sense promoter whereas the expression under the V-sense promoter was very weak in the absence of the transactivator C2. Untransformed N. benthamiana plants were agroinfiltrated with binary vector constructs containing V-sense-GFP alone or along with C1, C2, C4, V1, V2 or betaC1 (in both sense and antisense orientations) to understand the roles of these gene products in transactivation and/or suppression of post-transcriptional gene silencing (PTGS). The results showed strong suppression of gene silencing activities for C4 and betaC1 but a weak activity for C2. The suppression activities were also confirmed using gfp-silenced GFP16c/GFPi plants by agroinfiltration and agroinoculation. The expression of C4 and betaC1 as transgenes produced abnormal phenotypic growth compared to the other viral genes mentioned above, further supporting their suppressor function.