ScV is a simple dsRNA virus with two, separately encapsidated dsRNAs. Like the fungal viruses, ScV exhibits only a latent (or persistent) infection. The larger viral dsRNA (L) encodes the major capsid protein. The smaller (M) encodes a toxin (""""""""killer factor"""""""") lethal to strains without ScV-M particles. L and M have no detectable sequence homology and ScV-M particles are dependent on ScV-L particles for the synthesis of their capsid polypeptide and possibly for the synthesis of replicase and transcriptase polypeptides. Deletion mutations of M result in defective-interfering particles containing fragments of M (ScV-S particles). We want to establish the exact relation of the S RNAs to each other and to determine the sequences at the """"""""break points"""""""" where they originated by internal deletion of M. We will complete the 3' end sequences of the dsRNAs not yet entirely established using rapid gel sequencing methods on 3' T1 oligonucleotides. We will use the new RNAse H method to establish a fragment map of all the dsRNAs to extend the 3' end sequences and to generate 5' end sequences of the dsRNAs, to isolate for sequencing the break points of the DI RNAs, and to do a sequence comparison of selected regions of the DI RNAs. We will determine the relationship between the ScV-L transcript and its template by sequencing the 5' end of the transcript and comparing it to the 3' ends of L determined as above. Possible ribosome binding sites on the ScV-L transcript will be determined by RNAse protection experiments. Finally, we intend to clone ds cDNA made from the dsRNAs in order to size and purify ScV mRNAs, to determine the strandedness of the ScV RNAP transcripts, and to determine the extent of sequence heterogeneity in each ScV dsRNA.
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