All ETDs from UAB

Advisory Committee Chair

Casey Morrow

Advisory Committee Members

Jeffrey Engler

Peter Prevelige Jr

Stuart Frank

Wayne Sullender

Document Type


Date of Award


Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine


Reverse transcription is the hallmark of retroviruses, such as HIV-1. A cellular tRNA bound to the primer binding site region (PBS) is used for initiation of reverse transcription. HIV-1 specially selects tRNALys3 as primer, although changing the PBS can force HIV-1 to use alternative tRNAs as a primer. However, HIV-1 reverts back to use tRNALys3 following in vitro replication. The A-loop, located upstream of PBS, interacts with the anti-codon of primer tRNA. The inclusion of A-loop modification has been shown to stabilize the utilization of some alternative tRNAs. The studies in this dissertation focus on further defining the role of A-loop on primer selection To determine if certain tRNAs are favored by HIV-1 as primer, a series of mutants were constructed with PBS complementary to tRNAThr, tRNASer, tRNAPhe and tRNATyr. The corresponding mutants with additional A-loop modification were also constructed to investigate the importance of A-loop on the utilization of primer tRNA. Virus with only the PBS paired with tRNAThr, tRNASer and tRNAPhe reverted back to use tRNALys3, while viruses with tRNATyr PBS had severely compromised infectivity. The Aloop modification stabilized the use of tRNAThr but had no effect on the stable use of tRNASer. The A-loop mutation severely impaired the replication of virus with tRNAPhe PBS; the A-loop modification could not rescue the virus with tRNATyr PBS. Thus, the forced selection of certain alternative primers can have different effects on HIV-1 replication and PBS stability. To clarify the mechanism of how the A-loop influences primer iii selection, the A-loop region of virus with tRNAHis PBS was substituted with different Aloops complementary to the anti-codon of characterized tRNAs. Most of viruses with tRNAHis PBS maintained this PBS stably except virus with A-loop complementary to tRNASer. RNA modeling of A-loop revealed all had the potential to pair with the anticodon of tRNAHis, while the virus with tRNASer A-loop could not interact with the anticodon of tRNAHis. The results indicate that the A-loop could facilitate the utilization of tRNAHis as primer by attracting this tRNA to the PBS. Collectively, these results demonstrate that the PBS and A-loop are important determinants for primer selection. Complementarity between A-loop and tRNAHis is necessary for tRNAHis utilization as a primer.



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