Studies on Replication of Cucumber Mosaic Virus Satellite RNA
Satellite RNA variant-Q associated with cucumber mosaic virus (CMV) is a highly structured single-stranded RNA molecule of 336 nucleotides in length. Satellite RNA is an important subviral pathogen in agriculture because it modulates symptom expression in CMV-infected plants. In the agricultural industry, CMV is economically very important since it exhibits a broad host range comprising over 1200 plant species world-wide. Although satellite RNA has no sequence homology with CMV genome, satellite RNA replication is thought to occur entirely in the cytoplasm and is exclusively dependent upon CMV replicase. Chapter 1 of this dissertation describes an Agrobacterium-mediated transient gene expression system that allows the expression of individual viral and satellite RNA uncoupled from the virus replication in vivo, and by utilizing this system we demonstrate that CMV coat protein expressed independently of replication can enhance the accumulation of an individual CMV RNA. Virions formed by the CMV coat protein in the absence of virus replication encapsidates some host RNAs, and the satellite RNA can accumulate to a detectable level in the absence of the helper CMV. Chapter 2 describes a novel finding that satRNA expressed in the absence of its helper CMV is amplified by the host. By using immunostaining with antibody against double strand RNA, and confocal microscopy to identify the subcellular distribution of double stranded satellite RNA in situ, we have found that in the absence of the helper virus, satRNA can amplify in the nucleus. Furthermore, sequence analysis of satRNA oligomers formed in the absence of CMV replication showed that the junction between two monomeric forms have a unique heptanucleotide sequence GGGAAAA, which is not present in the junction of satRNA oligomers formed in the presence of CMV. Finally in chapter 3, by using agroinfiltration system, we demonstrate that the negative-sense strand of satellite RNA is not amplified by the host cell because it is not recruited into the nucleus. However, consistent with the previously published results, the negative-sense satellite RNA is replicated by the helper CMV but only to a low-level. These novel findings will place the viroids, hepatitis delta virus and satellite RNAs in a closer relationship with each other in terms of their replication mechanisms. Implications of these findings are discussed in Conclusion chapter of this dissertation.