Characterization of Tombusvirus-Like Associated RNAs and Investigation of a Subgenomic RNA Produced During Infections

Viral co-infections within a single host plant occur frequently in nature, with outcomes ranging from synergism to antagonism for the viruses involved. Autonomously replicating RNAs were found in co-infections with members of the genus Polerovirus and were found to depend upon the polerovirus for encapsidation, systemic movement, and aphid transmission. This thesis reports the identification of additional associated RNAs and phylogenetic and molecular characterization of their genomes. The associated RNAs were found to cluster as a monophyletic group composed of three subclades, and to be most closely related to the family Tombusviridae, thus they are hereafter referred to as tombusvirus-like associated RNAs (tlaRNAs). The capacity for autonomous replication was recapitulated for additional tlaRNAs representing two of the three subclades. Synergism was observed previously during co-infection of a tlaRNA designated ST9 and the polerovirus beet western yellows virus, prompting investigation into the tlaRNA genome and infection cycle. TlaRNAs produce two confirmed proteins during infection which constitute the RNA dependent RNA polymerase that enables their replication, though there is a small open reading frame (ORF) in the 3’ portion of the genome that is also present on a subgenomic RNA (sgRNA) generated during infection. This ORF was investigated for its protein coding potential, and the sgRNA for its potential to be a messenger RNA. Purification and analysis of polyribosomes, as well as mutational analyses strongly suggest the ORF is a sequence artifact and the sgRNA is not a messenger RNA. The sgRNA produced during infection of tlaRNA ST9 was determined to be about 470 nucleotides long and is coterminal with the genomic RNA 3’ end. Two hypotheses were explored in investigation of the mechanism of generation of this sgRNA, internal initiation of transcription from a subgenomic promoter and incomplete degradation of the genomic RNA by a host enzyme. Mutational analyses consisting of point mutations and larger deletions around the 5’ end of the sgRNA in the genomic context had no effect on accumulation of the sgRNA, thus use of a subgenomic promoter is an unlikely method for production of the sgRNA. Next, in vitro degradation assays using the exonuclease XRN1 were conducted with the 5’ portion of the 3’ UTR of ST9. These assays succeeded in demonstrating that a sequence within the ST9 3’ UTR is capable of resisting XRN1 mediated decay, and roughly mapped this sequence to near the 5’ end of the sgRNA. The results of the in vitro assay imply that the 3’ UTR of ST9 is capable of resisting degradation by the plant homolog of XRN1, XRN4, within the host. These results show that the sgRNA could be generated via the incomplete degradation of the genomic RNA by XRN4. Further experiments are needed to demonstrate this in planta and to confirm whether an additional method for production of the sgRNA exists. This thesis characterizes in greater detail the genome of tlaRNAs and suggests the sgRNA generated during infections is a noncoding RNA that is at least partially produced by the host decay machinery