The 3’ Utr of Turnip Crinkle Virus Interacts Locally and Distally to Regulate Transcription and Translation of the Virus

Title of dissertation: THE 3’ UTR OF TURNIP CRINKLE VIRUS INTERACTS LOCALLY AND DISTALLY TO REGULATE TRANSCRIPTION AND TRANSLATION OF THE VIRUS Megan Y. L. Young, Doctor of Philosophy, 2012 Dissertation Directed by: Professor Anne E. Simon Department of Cell Biology and Molecular Genetics Turnip crinkle virus (TCV) is a 4054 b positive-strand RNA virus of the genus Carmovirus in the Family Tombusviridae. Upon entry into cells, TCV is translated using host translational machinery to produce its RNA-dependent RNA polymerase (RdRp). The RNA is proposed to undergo a conformational rearrangement, mediated by recruitment of the RdRp to the 3’ ends of the viral RNA, which represses translation and promotes negative-strand synthesis. A second RNA switch is proposed to occur that inhibits minus-strand synthesis and promotes recruitment of the RdRp to the 3’ ends of negative-strands for the asymmetrical production of positive-strands. Within the 3’ UTR of TCV is a tRNA-shaped structure (TSS) that is capable of binding ribosomes and overlaps with structures necessary for translational enhancement. The RdRp has been shown to bind within this region and result in a widespread conformational shift. The binding of RdRp to the 3’ end of the virus is very sensitive to perturbations of sequence or structure, with many mutations resulting in non-specific binding of the RdRp. The elements within the 3’ UTR have been shown to be very interactive with alterations affecting the structure of regions hundreds of bases away. A second-site mutation study indicated that regions upstream of the 3’ UTR may also be interacting with the 3’ UTR. Some second-site mutations located in this upstream region were found to increase accumulation in protoplasts and additional studies are under way to explain this phenomenon. The 3’ viral contribution in a luciferase reporter construct was increased to incorporate the second-site mutations. While the second-site mutations had little effect on translation, it was surprising to find that extension of the viral 3’ sequence enhanced translation. Translational enhancement was mapped to just an additional twenty bases and further study revealed that a hairpin (H3) is important for viral translation and accumulation and may also be interacting with the 3’ UTR. THE 3’ UTR OF TURNIP CRINKLE VIRUS INTERACTS LOCALLY AND DISTALLY TO REGULATE TRANSCRIPTION AND TRANSLATION OF THE VIRUS by Megan Y. L. Young Dissertation Submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2012 Advisory Committee: Professor Anne E. Simon, Chair Professor Jonathan D. Dinman Professor James N. Culver Professor Brenda L. Fredericksen Professor Douglas A. Julin © Copyright by Megan Y. L. Young 2012 All rights reserved