| 摘要 |
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.
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