Sub-cellular localization of the grapevine rupestris stem pitting-associated virus replicase
The replicase polyprotein of 'Grapevine rupestris stem pitting-associated virus' (GRSPaV) was expressed in 'Nicotiana tabacum' cv. Bright Yellow-2 cells and found to form punctate structures in the cytoplasm, which were hypothesized to represent viral replication vesicles. A region within the methyltransferase (MTR) domain was found to be responsible for the formation of punctate structures that aligned along actin microfilaments, but did not associate with the ER, Golgi stacks, or peroxisomes. An arginine residue (R173) and a tryptophan residue (W184) within the MTR domain were tested as putative critical residues required for membrane association based on the formation of the observed puncta; however, replacement of these residues had no effect on punctal formation. The RNA-dependent-RNA-polymerase domain of the replicase was found to form aggregate structures in the cytoplasm when expressed alone, but colocalized with the puncta formed by the MTR during co-expression experiments, suggesting a possible interaction between these two proteins.