Studies of the requirements for the introduction of synthetic genes into reovirus.

Abstract

The purpose of this study was to introduce a synthetic reoviral gene into the reovirus particle. The gene was introduced in the form of (+) sense ssRNA transcripts. Reovirus type I infected cells were transfected with a synthetic deleted version of the T3 M1 gene of reovirus. Alternatively virions and cores modified by various chemical and physical treatments were incubated with synthetic (+) sense ssRNA copies of the M1 gene in replication assays. Neither of these methods resulted in successful replication of a synthetic gene or its introduction into reovirus. Studies examining the fate of transcripts showed that synthetic gene or its introduction into reovirus. Synthetic RNA was not recognized by viral proteins or particles when transfected into reovirus infected cells. Efforts were made to select lethal M1 mutants by passage of T1, T3 and G2 reovirus in $\mu$2 protein (product of M1 gene) expressing cells. These mutants could be used later as a selection system for reoviruses which rescued a synthetic M1 gene. In this system only reovirus with the rescued synthetic M1 gene should be able to grow on normal L929 cells. However, lethal M1 mutants were not obtained. Reovirus particles that had been subjected to physical and chemical treatments were analyzed by RNase A digestion and electron microscopy and by transcription assay. Modified core particles maintained or increased transcription activity relative to native cores. The most significant finding was the demonstration of active soluble transcriptase derived from viral cores that, previously, were thought to be the smallest component of the virus exhibiting transcriptase activity. This transcriptase complex is composed of dsRNA and $\lambda$3 protein. The problem of restricted access of exogenous RNA to the transcriptase can be overcome using this form of transcriptase and may be applicable to introduction of a synthetic gene into reovirus. (Abstract shortened by UMI.)