Functional Replacement of a Domain in the Rubella Virus P150 Replicase Protein by the Virus Capsid Protein

Abstract

The rubella virus (RUBV) capsid (C) protein rescues mutants with a lethal deletion between two in-frame NotI sites in the P150 replicase gene, a deletion encompassing nucleotides 1685 to 2192 of the RUBV genome and amino acids (aa) 548 to 717 of P150 (which has a total length of 1,301 aa). The complete domain rescuable by the C protein was mapped to aa 497 to 803 of P150. Introduction of aa 1 to 277 of the C protein (lacking the C-terminal E2 signal sequence) between the NotI sites in the P150 gene in a replicon construct yielded a viable construct that synthesized viral RNA with wild-type kinetics, indicating that C and this region of P150 share a common function. Further genetic analysis revealed that an arginine-rich motif between aa 60 and 68 of the C protein was necessary for the rescue of ⌬NotI deletion mutants and substituted for an arginine-rich motif between aa 731 and 735 of the P150 protein when the C protein was introduced into P150. Possible common functions shared by these arginine-rich motifs include RNA binding and interaction with cell proteins.

Determinants in the Maturation of Rubella Virus P200 Replicase Polyprotein Precursor

“…While it is recognized that large-scale deletions can have large effects on secondary structure, this type of mapping was successfully used to identify three alpha helices (one of them is the focus of the current study) in P150 that contribute to fiber formation (29,30) and the ⌬NotI domain (42,43). In both cases, finer-tuned mapping has been conducted to reveal valuable insight into those domain functions, and the current study aids in extending those findings.…”

“…The RUBV CP rescues lethal internal deletions of the Q domain in P150 (42) at an early stage of RUBV replication (i.e., before the generation of RCs or accumulation of viral RNAs), suggesting that it may influence a step in P200 maturation. In fact, the Q domain can be functionally replaced by CP insertion (43). The series of Robo502 constructs harboring the alanine-scanning substitutions between aa 36 and 49 made ideal constructs to test the effect of CP on P200 maturation, since most of these mutations (with the exceptions of T42A and Q45A) were lethal by the criterion that the mutants failed to synthesize detectable CP by 72 h posttransfection (data not shown).…”

“…While serving as an mRNA for the structural proteins appears to be the only role of the subgenomic RNA, newly synthesized genomic RNAs subsequently either undergo translation, producing P200 to recapitulate RC assembly and RNA synthesis, or are packaged into virus particles. Besides its role in forming virus particles, CP performs several nonstructural functions during virus infection (15), the most intriguing of which is its ability to rescue lethal mutations in the Q domain (a proline and arginine-rich domain) of P150 as well as within the 5= and 3= cis-acting elements (CAEs) of the genomic RNA (42,43,45).…”

“…While serving as an mRNA for the structural proteins appears to be the only role of the subgenomic RNA, newly synthesized genomic RNAs subsequently either undergo translation, producing P200 to recapitulate RC assembly and RNA synthesis, or are packaged into virus particles. Besides its role in forming virus particles, CP performs several nonstructural functions during virus infection (15), the most intriguing of which is its ability to rescue lethal mutations in the Q domain (a proline and arginine-rich domain) of P150 as well as within the 5= and 3= cis-acting elements (CAEs) of the genomic RNA (42,43,45).The precise molecular events that take place during RUBV infection from the time of genomic RNA translation to RC assembly remain poorly understood. However, replicon RNA constructs in which the structural protein ORF is replaced with a reporter gene, i.e., green fluorescent protein (GFP), are able to replicate, although they do not routinely spread from cell to cell (41), demonstrating that only the NSPs are necessary for RC formation and RNA replication.…”

Analysis of base and codon usage by rubella virus

“…Within the RUBV NS-ORF, the HVR resides within the recently defined Q domain. The function of the Q domain is unknown, and it was mapped through the curious observation that while deletions of this region render RUBV constructs nonviable, these deletions can be rescued by the capsid protein [51]. The region encoded by the HVR is high in proline and includes PxxP and PxxPxR motifs known to be important in proteinprotein interaction [28].…”