Characterization of Toscana virus-defective interfering particles generated in vivo

Toscana (TOS) virus stocks strongly interfering with standard virus replication were obtained by sequential passages of virus in suckling mouse brain. Characterization of viral RNAs in these stocks showed the presence of a heterogeneous population of defective RNA molecules derived from the L genomic segment, in both nucleocapsid (NC) and messenger RNAs, suggesting that these molecules could be replicated, assembled, and transcribed. Subgenomic RNAs from the L segment but not from the S or M segments were found in cells infected with these stocks. Defective RNA molecules interfered with virus replication and retained 5' and 3' genomic termini. Nucleotide sequence analysis of some cloned defective interfering (DI) RNAs revealed they contained one or more internal deletions reducing their length to 7-13% of the full-length L segment. An identical sequence motif, of variable length, was found at both terminal sites of the RNA junction on standard L sequences. This motif was retained only in one copy in the subgenomic RNA. These results are consistent with the generation of TOS virus DI particles in vivo and suggest that the defective genomic RNAs could be generated by polymerase jumping from a sequence to an identical one spatially closed because of the RNA structure.