Recombinant negative strand RNA virus expression systems
US5840520A · kind A · utility
Assignee
Inventors
Key dates
| Filing date | Sep 30, 1994 |
| Grant date | Nov 24, 1998 |
| Priority date | — |
| Expiry date | Sep 30, 2014 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12N2840/20
- WIPO fieldPharmaceuticals
- WIPO sectorChemistry
Abstract
Recombinant negative strand virus RNA templates which may be used to express heterologous gene products and/or to construct chimeric viruses are described. Influenza viral polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicative that the promoter lay solely within the 15 nucleotide 3' terminus. Sequences not specific for the influenza viral termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus sense cRNA were copied at low levels. The specificity for recognition of the virus-sense promoter was further defined by site-specific mutagenesis. It was also found that increased level of viral protein were required in order to catalyze both the cap-endonuclease primed and primer-free RNA synthesis from these model templates as well as from genomic length RNAs. This indicated that this reconstituted system had catalytic properties very similar to those of native viral RNPs. High levels of expression of a heterologous gene was obtained using the constructs and meth…
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