Protein 2A of Grapevine Fanleaf Nepovirus Is Implicated in RNA2 Replication and Colocalizes to the Replication Site

RNA2 of grapevine fanleaf virus is replicated in trans by the RNA1-encoded replication machinery. Full processing of the RNA2-encoded polyprotein P2 yields protein 2A of unknown function, the movement protein 2BMP, and the coat protein 2CCP. Analysis of a set of deletion mutants in the P2-coding seq...

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Bibliographic Details
Published in:Virology (New York, N.Y.) N.Y.), 1999-11, Vol.264 (1), p.25-36
Main Authors: Gaire, Fabien, Schmitt, Corinne, Stussi-Garaud, Christiane, Pinck, Lothaire, Ritzenthaler, Christophe
Format: Article
Language:English
Subjects:
Base Sequence
Cloning, Molecular
DNA Primers
Grapevine fanleaf virus
Green Fluorescent Proteins
Life Sciences
Luminescent Proteins - genetics
Luminescent Proteins - metabolism
Molecular Sequence Data
Nepovirus - genetics
Nepovirus - physiology
Open Reading Frames
Protein Biosynthesis
Protoplasts - virology
Recombinant Fusion Proteins - metabolism
RNA, Viral - genetics
Rosales - virology
Transcription, Genetic
Transfection
viral proteins
Viral Proteins - genetics
Viral Proteins - metabolism
Virus Replication
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Summary:RNA2 of grapevine fanleaf virus is replicated in trans by the RNA1-encoded replication machinery. Full processing of the RNA2-encoded polyprotein P2 yields protein 2A of unknown function, the movement protein 2BMP, and the coat protein 2CCP. Analysis of a set of deletion mutants in the P2-coding sequence revealed that protein 2A is necessary but not sufficient for RNA2 replication. In addition to the 5′ and 3′ noncoding sequences and the 2A-coding sequence, an additional sequence coding for 2BMP and/or 2CCP or the green fluorescent protein (GFP) is necessary for RNA2 replication. When 2A fused to GFP (2AGFP) was transiently expressed in uninfected T-BY2 protoplasts, 2AGFP appeared as punctate structures evenly distributed in the cytoplasm. However, in cells cotransfected with grapevine fanleaf virus RNAs and the 2AGFP construct, 2AGFP was predominantly found in a juxtanuclear location along with 1Dpro and 1CVPg, two RNA1-encoded proteins involved in RNA replication. Viral RNA replication as traced by 5-bromouridine 5′ triphosphate (BrUTP) incorporation into newly synthesized RNA occurred at the same location. This colocalization is consistent with the hypothesis that 2A enables RNA2 replication through its association with the replication complex assembled from RNA1-encoded proteins.
ISSN:0042-6822
1096-0341
DOI:10.1006/viro.1999.9927