Advisory Committee Chair
Casey Morrow
Advisory Committee Members
Peter Burrows
Chaenbei Chang
Jim Collawn
Jeffrey Engler
Document Type
Dissertation
Date of Award
2007
Degree Name by School
Doctor of Philosophy (PhD) School of Health Professions
Abstract
The features of tRNALys,3 that dictate why human immunodeficiency virus exclu-sively selects this tRNA as the primer for initiation of reverse transcription is unknown. The post-transcriptional modification at nucleotide 58 in the TΨC stem-loop could play a role during plus-strand synthesis to stop reverse transcriptase from re-copying the tRNA primer. Nucleotides 53 and 54 within the TΨC stem loop of the tRNA have been shown to be important to form the complex between tRNA and the HIV-1 viral genome during initiation of reverse transcription. To further delineate the features of the TΨC stem loop in tRNALys,3 in reverse transcription, we used a complementation system in which E.coli tRNALys,3 is provided in trans to a mutated HIV-1 genome in which the PBS is comple-mentary to this tRNA to ascertain the effects that different mutants in the TΨC stem loop of tRNALys,3 have on complementation. Alteration of nucleotide 58 from A to U (A58U), T54G and TG5453CC all resulted in tRNALys,3 that was aminoacylated when expressed in cells, while a T54C mutation resulted in a tRNALys,3 that was not aminoacylated. Both the A58U and T54G mutated tRNALys,3 complemented HIV-1 replication similar to wild type E.coli tRNALys,3, but the TG5453CC tRNALys,3 mutant did not complement replica-tion, thus, post-transcriptional modification of nucleotide 58 in tRNALys,3 is not essential for HIV-1 reverse transcription. In contrast, nucleotides 53 and 54 of tRNALys,3 are im-portant for aminoacylation and selection and use of the tRNALys,3 in reverse transcription. iii My research also focused on the link between the process of primer selection and lysine codon use in Gag. Proviral genomes were created in which the five codons specif-ic for lysines prior to the Gag-pol junction were altered to be specific for tRNALys,3 or tRNALys1,2. Greater amounts of infectious virus were produced using the in trans com-plementation system in which the five codons were specific for tRNALys,3 compared to the wild type or genomes with the five codons specific for tRNALys1,2. To extend this work, viral genomes were created in which all of the lysine codons except the five lysine codons prior to the Gag-pol junction were modified to be specific for tRNALys1,2. Analy-sis of the production of infectious virus from transfection of these proviral genomes re-vealed lower levels of virus regardless of whether the five codons preceding the Gag-pol frameshift were specific for tRNALys,3 or tRNALys1,2. These results demonstrate that structure features of tRNALys,3 and lysine codon preference within the Gag protein of HIV-1 have profound impact on the production of infectious virus and lead support to the idea that there is a link between viral translation and the process by which HIV-1 selects the tRNA primer used for reverse transcription.
Recommended Citation
Yu, Wanfeng, "Importance Of tRNAlys,3 Structure And Use In Gag Translation For Primer Selection Required For Replication Of Human Immunodeficiency Virus Type I" (2007). All ETDs from UAB. 3832.
https://digitalcommons.library.uab.edu/etd-collection/3832
