Patent ID: 6943015

Claim:
A method of producing packaged alphavirus particles, comprising the steps of: (a) transfecting a first set of cells with (i) a Sindbis virus replicon comprising a sequence encoding a nonstructural protein nsp2 that comprises an amino acid selected from the group consisting of proline, leucine, glycine and valine at amino acid position 726; (ii) a first helper RNA comprising a sequence encoding an alphavirus capsid protein and cis-acting elements that allow efficient replication and packaging of said first helper RNA and (iii) a second helper RNA comprising a sequence encoding the alphavirus glycoproteins E1 and E2 and cis-acting elements that allow efficient replication and packaging of said second helper RNA; (b) obtaining a primary stock of viral particles comprising (i) viral particles containing said Sindbis virus replicon, (ii) viral particles containing said first helper RNA, and (iii) viral particles containing said second helper RNA; (c) infecting a second group of cells with said primary stock of viral particles at high multiplicity of infection; and (d) obtaining a larger secondary stock of packaged viral particles comprising (i) viral particles containing said Sindbis virus replicon, (ii) viral particles containing said first helper RNA, and (iii) viral particles containing said second helper RNA, wherein said secondary stock has a titer of packaged replicons at least 1×10 8 infectious units/ml.