Date of Award

Summer 1994

Document Type


Degree Name

Doctor of Philosophy (PhD)


Chemistry and Biochemistry


Biomedical Sciences

Committee Director

Laura K. Moen

Committee Member

Frank Castora

Committee Member

Christopher Osgood

Committee Member

Mark S. Elliot


Human Immunodeficiency Virus, type 1 (HIV-1), is the causative agent of the Acquired Immunodeficiency Syndrome (AIDS). HIV-1 reverse transcriptase (RT), a heterodimer p66/p51, has been the major target for treatment of AIDS. The significance of the p51 subunit and the RNase H domain of p66 in terms of their influence on the RNA-dependent DNA synthesis was investigated. Clones of the wildtype HIV-1 RT subunits, p66 and p51, and a recombinant C-terminal deletion mutant, p64, [Barr, P. J. (1987) Bio/Technoloav 5, 486-489] were employed to study the structure-substrate binding relationships of HIV-1 RT. The activity assays of RNA-dependent DNA synthesis on both poly(rA)(dT) and a random base RNA template hybridized with a DNA oligomer showed that p51 significantly affects the enzyme activity. The increase in processivity by p51 in the p66/p51 heterodimer was also demonstrated. These observations suggested that the integrity of p51 is important in subunit-interactions for maintaining a favorable conformation of the enzyme for optimal function. C-terminal deletion in p66 was seen to decrease the processivity. The dissociation constant (Kd) for poly(rA)(dT) obtained by nitrocellulose binding assays suggested that the processivity of HIV

1 RT on poly(rA)(dT) correlated with the affinity for the substrate. The processivity of RT on RNA335-DNA20 was seen to be affected by the pause sites observed on the autoradiograms. The pauses of DNA synthesis tended to occur at positions of template containing poly G-C sequences. The order of processivity observed on RNA335-DNA20 was p64/p64, p66/p66 < p64/p51 < p66/p51. The C-terminal deletion in p66 was shown to affect the ability to extend the DNA strand on RNA template. In those non-wildtype forms of HIV-1 RT (p66/p66, p64/p64, and p64/p51), the affinity for primer-template seemed to be sensitive to the structure of the RNA template as seen when comparing Kds between poly(rA)(dT) and RNA335-DNA20. The wildtype enzyme, p66/p51, appeared to have a similar affinity for both substrates.