Molecular analysis of defective interfering RNAs associated with cucumber necrosis virus infections

In this thesis, the molecular biology of defective interfering (DI) RNAs associated with cucumber necrosis virus (CNV), a simple, well characterized plant RNA virus, was examined to gain insight into CNV RNA replication. Seven different cDNA clones of DI RNAs associated with either a laboratory strain of CNV or nucleic acid extracts from serially-passaged CNV infections (de now-generated DI RNAs) were constructed using reverse transcription followed by the polymerase chain reaction (RT-PCR). The sequence of each clone was determined and RNA transcripts generated by T7 RNA polymerase were assessed for biological activity by coinfecting with CNV. This analysis demonstrated that three or four large sequence blocks of the CNV genome are retained in CNV DI RNAs. These sequence blocks therefore likely represent essential c/s-acting elements involved in viral RNA replication. The presence of DI RNAs in CNV infected plants interfered with the development of the severe necrosis typical of CNV infections. Total nucleic acid extracts from coinfections were also found to contain RNAs which were twice the size of the DI RNA used in the coinfection. Using RT-PCR analyses and direct sequencing of RNA templates, these RNAs were established to be linear, head-to-tail repeats of DI RNA sequence, the first described in association with tombusvirus DI RNAs. Further characterization revealed that DI RNA dimers are likely generated from monomers by RNA recombination. Individual DI RNAs were found to vary in the amount of dimer which they accumulate during coinfection. Dimer accumulation was examined by coinfecting a series of chimeric and mutated DI RNAs constructed from a DI RNA which accumulated primarily as monomer and one which accumulated primarily as dimer. These analyses identified two distinct tracts of sequence in the 3' terminal region which correlate with increased dimer accumulation. The results of these investigations are discussed in terms of the insights they provide into CNV replication and recombination. A model for the generation of DI RNA dimers is also proposed.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat