title: Determinants in the maturation of rubella virus p200 replicase polyprotein precursor.
abstract: Rubella virus (RUBV), a positive-strand RNA virus, replicates its RNA within membrane-associated replication complexes (RCs) in the cytoplasm of infected cells. RNA synthesis is mediated by the nonstructural proteins (NSPs) P200 and its cleavage products, P150 and P90 (N and C terminal within P200, respectively), which are processed by a protease residing at the C terminus of P150. In this study of NSP maturation, we found that early NSP localization into foci appeared to target the membranes of the endoplasmic reticulum. During maturation, P150 and P90 likely interact within the context of P200 and remain in a complex after cleavage. We found that P150-P90 interactions were blocked by mutational disruption of an alpha helix at the N terminus (amino acids [aa] 36 to 49) of P200 and that these mutations also had an effect on NSP targeting, processing, and membrane association. While the P150-P90 interaction also required residues 1700 to 1900 within P90, focus formation required the entire RNA-dependent RNA polymerase (aa 1700 to 2116). Surprisingly, the RUBV capsid protein (CP) rescued RNA synthesis by several alanine-scanning mutations in the N-terminal alpha helix, and packaged replicon assays showed that rescue could be mediated by CP in the virus particle. We hypothesize that CP rescues these mutations as well as internal deletions of the Q domain within P150 and mutations in the 5' and 3' cis-acting elements in the genomic RNA by chaperoning the maturation of P200. CP's ability to properly target the otherwise aggregated plasmid-expressed P200 provides support for this hypothesis.
authors.affiliation: Georgia State University, Biology Department, Atlanta, Georgia, USA.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Matthews
authors.forename: Wen-Pin
authors.initials: WP
authors.lastname: Tzeng
authors.forename: Teryl K
authors.initials: TK
authors.lastname: Frey
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D020815:Polyproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012409:Rubella / Q000821:virology*; D012412:Rubella virus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D020815:Polyproteins; D011498:Protein Precursors; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase