ABSTRACT Title of dissertation: DIFFERENT REPLICATION REQUIREMENTS IN THE HOMOLOGOUS 3' ENDS OF A POSITIVE STRAND RNA VIRUS AND ITS SUBVIRAL RNA

Title of dissertation: DIFFERENT REPLICATION REQUIREMENTS IN THE HOMOLOGOUS 3' ENDS OF A POSITIVE STRAND RNA VIRUS AND ITS SUBVIRAL RNA Rong Guo, Doctor of Philosophy, 2010 Dissertation directed by: Professor Anne E. Simon Department of Cell Biology and Molecular Genetics SatC is a noncoding subviral RNA associated with Turnip Crinkle Virus (TCV), a small (4054 nt) single-stranded (+)-strand RNA virus belonging to the Carmovirus genus. Because of its small size (356 nt) and TCV-derived 3' end, satC has been successfully used as a model to elucidate sequence and structural requirements for TCV RNA replication. Although satC is considered a model to identify cis-acting elements required for TCV replication, recent findings indicate distinct differences in structures and functions of these related sequences. RNA2D3D predicts that part of the TCV 3' end (H5, H4a, H4b and two pseudoknots) folds into an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits and is required for translation. SatC contains a similar 3' end with 6 nt differences in the 100 nt TSS region. RNA2D3D did not predict a similar structure for satC TSS region, and satC did not bind yeast ribosomes. satC nucleotides were changed into TCV TSS bases to determine which base differences are responsible for the loss of the TSS in satC. Changing these bases all increased ribosome binding but surprisingly none of them had an effect on satC accumulation in protoplasts and plants. Therefore satC may need these and other 3' end base differences for its required conformational switch for efficient replication, and not to inhibit ribosome binding. In vivo genetic selection (SELEX) of the linker sequence between H5 and the Pr showed the conservation of UCC, which led to the discovery of Ψ2. Ψ2 is required for both viral and satC accumulation in protoplasts. H5-Pr linker had no significant structural change after RdRp binding in satC, which is different with TCV H5-Pr linker. TCV H5Pr linker had a major structural change upon RdRp binding, and is proposed to be involved in a conformational switch. Replacement of satC H4a with randomized sequence and scoring for fitness in plants by SELEX resulted in winning sequences that contain an H4a-like stem-loop. SELEX of H4a/H4b in satC generated two different structures: wt H4a/H4b-like structure and a single hairpin structure. Two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts. With the protection of CP, satC can form higher amount of dimers that have additional nucleotides at the junction sites in the absence of TCV. The extra nucleotides are not necessarily associated with an active TCV RdRp. DIFFERENT REPLICATION REQUIREMENTS IN THE HOMOLOGOUS 3' ENDS OF A POSITIVE STRAND RNA VIRUS AND ITS SUBVIRAL RNA