Recombinant negative strand RNA virus expression-systems

Details Recombinant negative strand RNA virus expression-systems Recombinant negative strand RNA virus expression-systems

1990-05-22

1992-11-24

Schwartz, Richard A.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

435 91, 4351723, 435194, 4352351, 4353201, 536 27, 935 32, 935 34, 935 57, C12P 2100, C12N 1500, C12N 1585, C12N 701

Patent

active

051660571

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 levels 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 lengths 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 methods described.

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This invention was funded in part by a grant from the National Institutes of Health, and the Government has certain rights in the invention.

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