Complementation of a Deletion in the Rubella Virus P150 Nonstructural Protein by the Viral Capsid Protein

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Abstract
Rubella virus (RUB) replicons with an in-frame deletion of 507 nucleotides between two Not I sites in the P150 nonstructural protein (Δ Not I) 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). Surprisingly, virus with Δ Not I was viable, and it was hypothesized that this was due to complementation of the Not I deletion by one of the virion structural protein genes. Introduction of the capsid (C) protein gene into Δ Not I-containing replicons as an in-frame fusion with a reporter gene or cotransfection with both Δ Not I replicons and RUB replicon or plasmid constructs containing the C gene resulted in replication of the Δ Not I 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 . Approximately the 5′ one-third of the C gene was necessary for complementation. 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. Complementation occurred at a basic step in the virus replication cycle, because Δ Not I replicons failed to accumulate detectable virus-specific RNA.