![]() Recently, we developed a RUB replicon (RUBrep) in which the SP-ORF was replaced with a reporter gene, such as that coding for chloramphenical acetyltransferase (CAT) or green fluorescent protein (GFP) ( 32). The 3′-proximal ORF, or structural protein ORF (SP-ORF), which is translated from a subgenomic (SG) RNA, encodes the virion proteins in the order 5′-C-E2-E1-3′ processing of these proteins is mediated by the cellular enzyme signal endopeptidase. The 5′-proximal ORF, or nonstructural protein ORF (NS-ORF), is translated from the genome RNA into a 240-kDa precursor that is proteolytically cleaved at a single site by a virus-encoded protease into two products: an N-terminal product of 150 kDa (P150) and a C-terminal product of 90 kDa (P90). The RUB genome is 9,762 uncleotides (nt) in length, of positive-polarity, and contains two long open reading frames (ORFs). ![]() The RUB virion consists of a genomic, single-stranded RNA enclosed in a quasispherical capsid composed of multiple copies of the viral capsid protein, C, which is in turn surrounded by a lipid bilayer envelope in which are embedded two virus glycoproteins, E1 and E2. Rubella virus (RUB) is the sole member of the genus Rubivirus in the family Togaviridae (for a review, see reference 7). Complementation occurred at a basic step in the virus replication cycle, because Δ NotI replicons failed to accumulate detectable virus-specific RNA. Mutations that prevented translation of the C protein while minimally disturbing the C gene sequence abrogated complementation, while synonymous codon mutations that changed the C gene sequence without affecting the amino acid sequence at the 5′ end of the C gene had no effect on complementation, indicating that the C protein, not the C gene RNA, was the moiety responsible for complementation. Approximately the 5′ one-third of the C gene was necessary for complementation. Introduction of the capsid (C) protein gene into Δ NotI-containing replicons as an in-frame fusion with a reporter gene or cotransfection with both Δ NotI replicons and RUB replicon or plasmid constructs containing the C gene resulted in replication of the Δ NotI replicon, confirming the hypothesis that the C gene was the structural protein gene responsible for complementation and demonstrating that complementation could occur either in cis or in trans. Surprisingly, virus with Δ NotI was viable, and it was hypothesized that this was due to complementation of the NotI deletion by one of the virion structural protein genes. Rubella virus (RUB) replicons with an in-frame deletion of 507 nucleotides between two NotI sites in the P150 nonstructural protein (Δ NotI) do not replicate (as detected by expression of a reporter gene encoded by the replicon) but can be amplified by wild-type helper virus (Tzeng et al., Virology 289:63-73, 2001).
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