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 Irans by the RNA1-encoded replication machinery. Full processing of the RNA2-encoded polyprotein P2 y ields protein 2A of unknown function, the movement protein 2B(MP),and the c oat protein 2C(CP). 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-co ding sequence, an additional sequence coding for 2B(MP) and/or 2C(CP) Or th e green fluorescent protein (GFP) is necessary for RNA2 replication. When 2 A fused to GFP (2AGFP) was transiently expressed in uninfected T-BY2 protop lasts, 2AGFP appeared as punctate structures evenly distributed in the cyto plasm. However, in cells cotransfected with grapevine fanleaf virus RNAs an d the 2AGFP construct, 2AGFP was predominantly found in a juxtanuclear loca tion along with 1D(pro) and 1C(VPg), two RNA1-encoded proteins involved in RNA replication. Viral RNA replication as traced by 5-bromouridine 5' triph osphate (BrUTP) incorporation into newly synthesized RNA occurred at the sa me location. This colocalization is consistent with the hypothesis that 2A enables RNA2 replication through its association with the replication compl ex assembled from RNA1-encoded proteins, (C) 1999 Academic Press.