Peanut clump virus RNA-1-Encoded p15 regulates viral RNA accumulation but is not abundant at viral RNA replication sites

RNA-1 of peanut clump pecluvirus (PCV) encodes N-terminally overlapping pro teins which contain helicase-like (P131) and polymerase-like (P191) domains and is able to replicate in the absence of RNA-2 in protoplasts of tobacco BY-2 cells. RNA-1 also encodes P15, which is expressed via a subgenomic RN A. To investigate the role of P15, we analyzed RNA accumulation in tobacco BY-2 protoplasts inoculated with RNA-1 containing mutations in P15. For all the mutants, the amount of progeny RNA-1 produced was significantly lower than that obtained for wild-type RNA-I. If RNA-2 was included in the inocul um, the accumulation of both progeny RNAs was diminished, but near-normal y ields of both could be recovered if the inoculum was supplemented with a sm all, chimeric viral replicon expressing P15, demonstrating that P15 has an effect on viral RNA accumulation. To further analyze the role of P15, trans cripts were produced expressing P15 fused to enhanced green fluorescent pro tein (EGFP). Following inoculation to protoplasts, epifluoresence microscop y revealed that P15 accumulated as spats around the nucleus and in the cyto plasm. Intracellular sites of viral RNA synthesis were visualized by laser scanning confocal microscopy of infected protoplasts labeled with 5-bromo-u ridine 5'-triphosphate (BrUTP), BrUTP labeling also occured in spats distri buted within the cytoplasm and around the nucleus. However, the BrUTP-label ed RNA and EGFP/P15 very rarely colocalized, suggesting that P15 does not a ct primarily at sites of viral replication but intervenes indirectly to con trol viral accumulation levels.