Advisory Committee Chair
Casey Morrow
Advisory Committee Members
Jeffrey Engler
Peter Burrows
David Ansardi
William Britt
Document Type
Dissertation
Date of Award
2007
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS). HIV replication includes the notable process of reverse transcription, a conversion of RNA to DNA. Reverse transcription is initiated from a primer by a viral enzyme, reverse transcriptase. The primer, tRNALys,3, is selected from the cytoplasm of an infected cell, annealed via the 3’ terminal 18-nucleotides to the viral primer binding site (PBS), and used in a subsequent infection. The mechanism of primer selection has not been determined although numerous studies have been performed. This has been hampered by the challenge of tRNALys,3 manipulation in the cell and dissection of the viral and cellular mechanisms without affecting the cell. To better understand primer selection, we have employed the use of an alterative tRNA primer, Escherichia coli tRNALys,3. An HIV-1 provirus was engineered to contain a PBS relevant to E. coli tRNALys,3. This HIV-1 provirus required co-transfection of the plasmid encoding E. coli tRNALys,3 for production of infectious virus. E. coli tRNALys,3 was expressed, aminoacylated, and selected by HIV-1 as a primer. Increasing E. coli tRNALys,3 plasmid concentrations directly influenced HIV-1 infectivity; however, the infectious virus levels were lower than wild type or in a system using yeast tRNAPhe. Additional E. coli tRNALys,3 mutants containing altered anticodon nucleotides had slightly varying results on HIV-1 infectivity, but highly varying results with aminoacylation, indicating that aminoacylation does not necessarily correlate with iii primer selection. Higher levels of infectious virus were achieved when the anticodon stem and the variable loop of E. coli tRNALys,3 were interchanged with mammalian tRNALys,3. Individual nucleotide changes in the stem region did not yield infectious virus levels comparable to that when all nucleotides were interchanged, indicating the requirement for all interchanged bases in the anticodon stem region for optimum tRNALys,3 usage as a primer. Collectively, our studies demonstrate that lysyl-tRNA synthetase (LysRS) is not the sole determinant of primer selection and usage, and interactions between the primer and the virus are highly intricate.
Recommended Citation
McCulley, Anna, "Primer Selection Of E. Coli tRNA Lys,3 By Human Immunodeficiency Virus Type-1" (2007). All ETDs from UAB. 3758.
https://digitalcommons.library.uab.edu/etd-collection/3758