Determinants of subcellular localization of the rubella virus nonstructural replicase proteins

Abstract

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.

Determinants in the Maturation of Rubella Virus P200 Replicase Polyprotein Precursor

“…Immunofluorescence was conducted as previously described (30), with some modifications. Briefly, Vero cells were grown on glass coverslips until 20 to 30% confluent and either infected with Robo502/P150-GFP or transfected with a plasmid(s) or in vitro transcripts.…”

“…The RUBV NSPs are initially distributed in a punctate pattern in the cytoplasm of infected cells (17,30). A unique NSP fiber network forms late during infection and was the subject of a previous communication (29).…”

“…Therefore, the effects of the C-terminal deletions on the localization of P200 were investigated using plasmid-based constructs that have a mutated protease cleavage site (designated by an asterisk). This mutation of 1299-GGG-1301 to 1299-VVV-1301 makes analysis of the uncleaved precursor possible, since without the mutation it would rapidly be processed (22,24,30). Upon expression (Fig.…”

“…The NSPs are initially translated as a polyprotein called P200 (5,9). P200 is thought to function in properly targeting the genomic RNA to initial sites of replication complex (RC) assembly where viral RNA synthesis occurs (30); however, little is known about the mechanisms by which this occurs. Subsequently, P200 functions in the synthesis of negative-strand RNA using the incoming genome as a template (23).…”

“…Early reports indicated that RUBV RCs form within invaginations on the outer membranes of lysosomes and late endocytic vesicles (6,20,27). However, recent examination of RUBV-infected cells revealed that P150 and double-stranded RNA (dsRNA; a standard marker for RCs) showed intracellular colocalization, but only marginally with late endosomes and lysosomes, as well as colocalization at the plasma membrane (30). Sindbis virus (SINV) and Semliki Forest virus (SFV), two other togaviruses belonging to the Alphavirus genus, though originally reported to be replicating their RNA in association with the endo-/ lysosomal compartment, have now been found to replicate in membranous spherules which originate at the plasma membrane and migrate to the perinuclear region via endocytosis (11)(12)(13).…”