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.

Short Self-Interacting N-Terminal Region of Rubella Virus Capsid Protein Is Essential for Cooperative Actions of Capsid and Nonstructural p150 Proteins

“…At 72 hpi, the majority of p150/AG1 was distributed in filamentous structures in the cytoplasm (Fig. 1D), as described previously for the original p150 (9,22,47). Almost all the p150/AG1 in these distributions clearly overlapped the distribution of the capsid protein ( Fig.…”

“…These data imply that the capsid protein is required for the initiation of viral RNA replication but not for its maintenance. p150 interacts with both p90 and the capsid protein (9,17,47), and p150 and p90 are thought to form the replication complex, together with the genomic RNA (8-10). The capsid protein can also bind the genomic RNA through amino acid residues 28 to 56 (48), immediately downstream from the putative coiledcoil domain.…”

Cellular responses to Sindbis virus infection of neural progenitors derived from human embryonic stem cells

“…Western Blot analysis was performed on cell lysates of hNPCs, either mock infected or SINV infected, at 4, 12, 24, 36, and 48 hours post infection, using protocols described previously [ 23 ]. Primary antibodies used were anti-GAPDH (1:5000; Abcam), anti-NF-kB p65 (1:200; Santa Cruz); anti-phospho-STAT3 (1:200; Cell signaling), anti-phospho-IRF3 (1:1000; Eptomics); anti-Nestin (1:500; Neuromics); anti-neuro-filament M (NF-M; 1:500; Neuromics); anti-Tuj1 (1:1000; Abcam); anti-PCNA (1:1000, Santa Cruz) and anti-cleaved-caspase 3 (1:1000; Cell Signaling).…”

Heat Shock Protein 90 Ensures the Integrity of Rubella Virus p150 Protein and Supports Viral Replication

“…Therefore, our interpretation of these data was that HSP90 activity is important for NSP processing. Both p90 and p150 were detected in these experiments when either p90-specific or FLAG-specific (p150-specifc) antibodies were used, because p90 and p150 interact with each other (36,37). This hypothesis was also assessed by a radiolabeling experiment using a p200 expression plasmid.…”