In vitro replication system capable of rescuing cloned and manip

Details In vitro replication system capable of rescuing cloned and manip In vitro replication system capable of rescuing cloned and manip

1994-06-01

1997-03-25

Guzo, David

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

435 9121, 435 9151, 435238, C12N 700, C12N 701, C12N 706, C12P 1934

Patent

active

056144032

ABSTRACT:
A method for template-dependent in vitro replication and transcapsidation of double stranded RNA from Reoviridae mRNA templates is described. This method provides an efficient means for isolating rotaviral mRNA, manipulating the mRNA, and expressing the new dsRNA species without the use of infected cells by constructing a new virus in the test tube without any cellular components. Viral protein complexes are prepared by test tube degradation of purified virus particles or by expression of a small subset of viral genes in insect cells, where the proteins form complexes. Viral mRNAs are made in the test tube by transcription of "native" viral mRNAs from viral transcriptase particles previously made in vitro, or by expression of viral mRNAs from transcription vectors in vitro. The resultant protein complexes and mRNAs are mixed with a combination of salts and substrates suitable for RNA synthesis, and the enzymatic activity inherent in the protein complexes results in the replication of the mRNA to produce dsRNA. Morphogenesis of the virus particle is completed by sequential transcapsidation with capsid proteins. Thus, the invention provides a method for building viruses with "custom made" genomes, allowing the practitioner to control and manipulate all components of the reaction, since the reaction contains only those components which the practitioner chooses to add. This constitutes the rescue of exogenous genes into rotavirus genomes. Rescue allows the powerful techniques of molecular biology to be applied to the study of viral gene function in the context of the virus-infected cell.

REFERENCES:
Fujimura et al, The Journal of Biological Chemistry, Jun. 26, 1989, vol. 264(18): pp. 10372-10377.

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