Determinants of subcellular localization of the rubella virus nonstructural replicase proteins

The rubella virus (RUBV) nonstructural replicase proteins (NSPs), P150 and P90, are proteolytically processed from a P200 precursor. To understand the NSPs' function in the establishment of virus RNA replication complexes (RCs), the NSPs were analyzed in virus-infected cells or cells transfected with NSP-expressing plasmids. In infected cells, P150 was localized in cytoplasmic foci at 24 hpi and in cytoplasmic fibers, unique to RUBV, by 48 hpi. RCs, marked by dsRNA, colocalized with P150-foci, but only occasionally with the endosome/lysosome marker LAMP-2, indicating that RNA synthesis occurs at other sites rather than exclusively in endosomes/lysosomes as was previously thought. An expressed cleavage-deficient form of P200 also localized to cytoplasmic foci, suggesting that the precursor is required for targeting to sites of RC establishment. P150 was found to be the determinant of fiber formation and the NSP membrane-binding domain was mapped to the N-terminus of P150.