P. Dunoyer et al., Peanut clump virus RNA-1-Encoded p15 regulates viral RNA accumulation but is not abundant at viral RNA replication sites, J VIROLOGY, 75(4), 2001, pp. 1941-1948
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.