Engineering the genome of Grapevine virus A into a vector for expression of proteins in herbaceous plants

• Published in: Journal of virological methods Vol. 132; no. 1; pp. 227 - 231
• Main Authors: Haviv, Sabrina, Galiakparov, Nurbol, Goszczynski, Dariusz E., Batuman, Ozgur, Czosnek, Henryk, Mawassi, Munir
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01-03-2006; Amsterdam Elsevier; New York, NY
• Subjects: Binding Sites; Biological and medical sciences; Capsid Proteins - biosynthesis; Capsid Proteins - genetics; Citrus tristeza virus; Cloning, Molecular; DNA Restriction Enzymes; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes, Reporter; Genetic Engineering; Genetic Vectors; Genome, Viral; Glucuronidase - biosynthesis; Glucuronidase - genetics; Grapevine virus A; Microbiology; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana - virology; Nicotiana benthamiana; Plant Viral Movement Proteins; Plant Viruses - genetics; Promoter Regions, Genetic; Recombinant Proteins - biosynthesis; RNA Viruses - genetics; Techniques used in virology; Viral Proteins - genetics; Virology; Virus-based expression vector; Vitaceae; Vitivirus; Virus-based expression vector; Grapevine virus A; Vitivirus; Virus; Microbiology; Flexiviridae; Method; Gene expression; Genome; Vector; Protein; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2005.10.020

Grapevine virus A (GVA), a species of the genus
Vitivirus, consists of a ∼7.4
kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). In addition to grape varieties, GVA infects
Nicotiana benthamiana plants and protoplasts. We engineered the genome of GVA as a vector that includes duplication of homologous sequences that contain the promoter of the movement protein (MP) sgRNA, supplemented by enzymatic restriction sites to be used as a convenient tool for transient expression of foreign genes from an individual sgRNA. The resulting vector was able to infect and to move in
N. benthamiana plants in a manner similar to the wild-type GVA, but it was not stable and the inserted sequence was lost from the genome. Replacing the duplicated promoter with a GVA-MP promoter derived from a distantly related isolate of GVA improved the stability of the inserted sequence. The resulting vector was successfully used to express the reporter gene beta-glucuronidase (GUS) and the coat protein gene of
Citrus tristeza virus in inoculated
N. benthamiana plants. Development of a useful GVA vector is expected to find a use as a biotechnological tool for improvement of grapevines and it may enable vine breeders to bypass obstacles involved in genetic manipulation of perennial and fruiting plants.